Recombinant bacterial cells for delivery of PNP to tumor cells

ABSTRACT

The present invention provides a procaryotic host cell stably transformed or transfected by a vector including a DNA sequence encoding for purine nucleoside phosphorylase or hydrolase. The transformed or transfected procaryotic host cell can be used in combination with a purine substrate to treat tumor cells and/or virally infected cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. Ser. No.09/183,188 filed Oct. 30, 1998, pending, which is a non-provisional ofprovisional 60/064,767 filed Oct. 31, 1997, now abandoned, and is also acontinuation-in-part application of U.S. Ser. No.08/881,772 filed Jun.24, 1997, now U.S. Pat. No. 6,017,896, which is a continuation-in-partapplication of U.S. Ser. No. 08/702,181 filed Aug. 23, 1996, nowabandoned, which is a continuation-in-part application of U.S. Ser. No.08/122,321 filed Sep. 14, 1993, now U.S. Pat. No. 5,552,311.

GRANT REFERENCE

[0002] The research carried out in connection with this invention wassupported in part by a grant from the National Cancer Institute (CA7763-02).

SEQUENCE LISTING

[0003] This application contains a Sequence Listing that is beingsubmitted herewith as a separate document.

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] This invention is in the field of cancer therapy and inparticular, relates to compositions and methods to specifically killtumor cells by the production of toxic compounds in the tumor cells.

[0006] 2. Description of the Related Art

[0007] Inefficiency of gene delivery, together with inadequate bystanderkilling, represent two major conceptual hurdles in the development of atoxin mediated gene therapy for human malignancy. Gene transfer is auseful adjunct in the development of new therapies for human malignancy.Tumor cell expression of histocompatibility antigens, cytokines, orgrowth factors (for example, IL-2, IL-4, GMCSF) appears to enhanceimmune-mediated clearance of malignant cells in animal models, andexpression of chemo-protectant gene products, such as p-glycoprotein inautologous bone marrow cells, is under study as a means of minimizingmarrow toxicity following administration of otherwise lethal doses ofchemotherapeutic agents.

[0008] Theoretically, the most direct mechanism for tumor cell killingusing gene transfer is the selective expression of cytotoxic geneproducts within tumor cells. However, no recombinant enzyme or toxin hasproven useful in mediating high levels of toxicity in unselected tumorcells. Classical enzymatic toxins such as pseudomonas exotoxin A,diphtheria toxin and ricin are unlikely to be useful in this context,since these enzymes kill only cells in which they are expressed, and nocurrently available gene transfer vector is capable of gene delivery toa sufficiently high percentage of tumor cells to make use of the aboverecombinant enzymes.

[0009] Another strategy that has been developed to selectively killtumor cells involves the delivery and expression of the HSV dThd kinasegene to replicating tumor cells followed by treatment with ganciclovir.Ganciclovir is readily phosphorylated by the HSV dThd kinase, and itsphosphorylated metabolites are toxic to the cell. Very littlephosphorylation of ganciclovir occurs in normal human cells. Althoughonly those cells expressing the HSV dThd kinase should be sensitive toganciclovir (since its phosphorylated metabolites do not readily crosscell membranes), in vitro and in vivo experiments have shown that agreater number of tumor cells are killed by ganciclovir treatment thanwould be expected based on the percentage of cells containing the HSVdThd kinase gene. This unexpected result has been termed the “bystandereffect” or “metabolic cooperation.” It is thought that thephosphorylated metabolites of ganciclovir may be passed from one cell toanother through gap junctions. However, even if a nucleosidemonophosphate such as ganciclovir monophosphate were released into themedium by cell lysis, the metabolite would not be able to enterneighboring cells and would likely be degraded (inactivated) to thenucleoside by phosphatases.

[0010] Although the bystander effect has been observed in initialexperiments using HSV dThd kinase, the limitations of current genedelivery vehicles mean that a much greater bystander effect is importantto successfully treat human tumors using this approach. One difficultywith the current bystander toxicity models is that bystander toxicitywith metabolites that do not readily cross the cell membrane will not besufficient to overcome a low efficiency of gene transfer (for example,transfection, transduction, etc.).

[0011] One protocol for treating brain tumors in humans uses retroviraldelivery of HSV dThd kinase, followed by ganciclovir administration. Inrat models, using HSV dThd in this context, tumor regressions have beenobserved. The HSV dThd kinase approach has not proven sufficient inhumans thus far; this may in part be due to (1) inadequate bystandertoxicity with HSV dThd kinase, and (2) cell killing only of dividingcells using HSV dThd kinase with ganciclovir. The usefulness of E. colicytosine deaminase, which converts 5-fluorocytosine to 5-fluorouracil,has recently been reported to provide substantial bystander toxicity.However, 5-FU is not a highly toxic compound in this setting andbystander killing in vitro has been inefficient, i.e., similar to thatof observed with HSV dThd kinase.

[0012] Prodrug activation by an otherwise non-toxic enzyme (for example,HSV dThd kinase, cytosine deaminase) has advantages over the expressionof directly toxic genes, such as ricin, diphtheria toxin, or pseudomonasexotoxin. These advantages include the capability to (1) titrate cellkilling, (2) optimize therapeutic index by adjusting either levels ofprodrug or of recombinant enzyme expression, and (3) interrupt toxicityby omitting administration of the prodrug. However, like otherrecombinant toxic genes, gene transfer of HSV dThd kinase followed bytreatment with ganciclovir is neither designed to kill bystander cellsnor likely to have broad bystander toxicity in vivo.

[0013] An additional problem with the use of the HSV dThd kinase orcytosine deaminase to create toxic metabolites in tumor cells is thefact that the agents activated by HSV dThd kinase (ganciclovir, etc.)and cytosine deaminase (5-fluorocytosine) kill only cells synthesizingDNA. Even if a considerable number of nontransfected cells are killed,one would not expect to kill the nondividing tumor cells with theseagents.

[0014] Thus, there exists a need for a toxin gene therapy method thatovercomes the problems of inefficient gene delivery, cellreplication-dependent killing and low toxin diffusion between cells. Thepresent invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

[0015] Accordingly to the present invention, a unique E. coli containingthe PNP gene (SEQ ID No: 5) is disclosed. This E. coli can be used totreat tumors in combination with a prodrug including MeP-dR. Also, amethod for causing tumor regression and/or inhibiting tumor growth isdisclosed which includes directly administering a purine analog to atumor.

[0016] In yet still another embodiment of the present invention, thereis provided a host cell transfected with the vector of the presentinvention which expresses a purine nucleoside phosphorylase protein.

[0017] Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification:It is to benoted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0019]FIG. 1 shows the toxicity due to DOTMA-DOPE liposomes used totransfect T-84 colon carcinoma cells with 10, 20 or 40 μg of cDNAcontaining either the E. coli PNP or LacZ genes under thetranscriptional control of SV-40 early promoter (SV-PNP and SV-LacZ,respectively) and the additional toxicity when MeP-dR (160 μM) is addedto T-84 transfected cells expressing the PNP gene (PNP+MeP-dR). Cellstransfected with SV-PNP construct were treated, with (PNP+MeP-dR) andwithout (PNP) MeP-dR. LacZ transfected cells were studied in the sameway. Nontransfected cells were treated with (MeP-dR) and without(control) MeP-dR.

[0020] FIGS. 2A-D show the human tyrosinase transcriptional promotersequence (Tyr)-restricted expression of the luciferase reporter gene(Luc), to which it was operable linked (Tyr-Luc), in melanoma cellsMel-1 and Mel-21 (FIG. 2A), and the SV40 early promoter (SV)constitutive expression of the Luciferase gene (Luc) to which it wasoperable linked (SV-Luc), in each carcinoma cell line (see FIGS. 2A-2D).Rev-Tyr-Luc, Tyr promoter sequence linked to the Luc gene in reverseorientation so that it does not transcribe Luc (no expression). Basic,promoterless Luc gene construct.

[0021]FIGS. 3A and 3B show the dependence of purine analog nucleosideMeP-dR toxicity on expression of E. coli purine analog nucleosidephosphorylase (PNP). SV-PNP, cells transfected with a construct in whichthe constitutive SV40 early promoter is operably linked to the PNP gene;Tyr-PNP, cells transfected with a construct, in which the melanomaspecific human tyrosinase promoter sequence is operably linked to PNPgene; Tyr-Luc, cells transfected with a construct in which the melanomaspecific human tyrosinase promoter sequence is operably linked toluciferase reporter gene; no-txf, cells not transfected with arecombinant construct. T-84, carcinoma cell line (3A); Mel-1, melanomacell line (3B).

[0022]FIG. 4 shows the difference in in vivo development of tumors inathymic nude mice engrafted with murine mammary carcinoma 16/C cellstransduced with the recombinant retroviral expression vector LN/PNP(which directs expression of E. coli PNP) depending on time ofadministration of MeP-dR prodrug. No injection of MeP-dR (control);injection of MeP-dR on days 1-4 post engraftment (early rx); injectionof MeP-dR on days 13-15 post engraftment (late rx) are shown.

[0023]FIG. 5 shows the effect of MeP-dR on transduced cells with stableE. coli PNP expression. FIGS. 5A and 5B show mixing experiments in whichthe transduced and wild type B16 (FIG. 5A) or 16/C (FIG. 5B) werecocultured. Complete abrogation of cellular proliferation was observedwhen as few as 2% of the cultured cells expressed E. coli PNP under theregulatory control of an SV-40 promoter. A high level bystander effectwas also observed when either B16 or 16/C cells expressed E. coli PNP,as measured by a standard cellular LDH release assay. Growthcharacteristics of transduced and wild type (nontransduced) B 16 cellswere identical in the absence of drug; the same was true of the wildtype and transduced 16/C cell lines.

[0024]FIG. 6 shows the effect of MeP-dR and F-araAMP on the growth ofwild-type 16/C tumors in animals. Both compounds had only a small effecton tumor growth. These results are in contrast with those in FIGS. 4 and7.

[0025]FIG. 7 shows the effect of F-araAMP on the growth of 16/C tumorsexpressing E. coli PNP. F-araAMP significantly inhibited the growth ofthese tumors. Contrast with the effect of F-araAMP on wild-type tumorsin FIG. 6.

[0026]FIGS. 8 and 9 show the effect of MeP-dR on the growth of wild-typeD54 tumors (FIG. 8) and E. coli PNP expressing D54 tumors (FIG. 9).These two figures are a graphical representation of the data shown inTable IV. FIG. 8 shows that MeP-dR did not affect parental D54 tumorcell growth. FIG. 9 shows that MeP-dR caused regression of D54 tumorsexpressing E. coli PNP. Note that in this figure tumors that completelyregressed are not included in the calculation of medium tumor weight.Therefore, since 4 animals had no tumors at the end of the experiment,the tumor weight on the days beyond day 40 refer to the two tumors thatdid not completely regress. In this experiment the two remaining tumorswere at the limit of detection and did not show any signs of growth pastday 30. Therefore, these animals may also be cured of their disease.

[0027]FIGS. 10 and 11 are a confirmation study of the experiment shownin FIGS. 8 and 9 that show the effect of MeP-dR on the growth ofwild-type D54 tumors (FIG. 10) and E. coli PNP expressing D54 tumors(FIG. 11). These two figures are a graphical representation of the datashown in Table V. FIG. 10 shows that MeP-dR at two doses did not affectparental D54 tumor cell growth. FIG. 11 shows that MeP-dR at both dosescaused regression of D54 tumors expressing E. coli PNP. Note that as inFIG. 9, tumors that completely regressed are not included in thecalculation of medium tumor weight. Therefore, since 4 animals, whichwere treated with 67 mg/kg MeP-dR, had no tumor at the end of theexperiment, the tumor weight on the days beyond day 40 refer to the sixtumors that did not completely regress.

[0028]FIG. 12 is a predicted restriction map of plasmid pTRCPNPcontaining the predicted 5013 base sequence pairs (SEQ ID No: 5)encoding E. coli PNP.

[0029]FIG. 13 illustrates the induction of E. coli PNP by IPTG.

[0030]FIG. 14 is a graph of median tumor weight versus the response ofSC Lewis Lung Tumors to treatment with MeP-dR and NSC 103543.

[0031]FIG. 15 is a histogram illustrating the tumor burden versus thetumor volume on day 16 following treatment with MeP.

[0032]FIG. 16 is a graph illustrating tumor burden over time followingtreatment with MeP.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The invention provides a method of killing replicating ornon-replicating, transfected or transduced mammalian cells and bystandercells, comprising the following steps: (a) transfecting or transducingtargeted mammalian cells with a nucleic acid encoding a suitable purinenucleoside cleavage enzyme which releases a purine analog from thesubstrate purine nucleoside or providing such enzyme directly to thetargeted cells; and (b) contacting the targeted cells expressing orprovided with the purine nucleoside cleavage enzyme with a substrate forthe enzyme to produce a toxic purine base thereby killing the targetedcells and also bystander cells not expressing or containing the cleavageenzyme. Thus, in the presence of substrate, the cleavage enzyme producesa toxic product. It should be appreciated that a “non-human or modifiedhuman purine analog nucleoside phosphorylase (PNP)” includes the use ofeither types of PNP in the same therapeutic regimen as the purinenucleoside cleavage enzyme. The killing can occur in vitro or in vivo.

[0034] In this method of the present invention, the targeted cells arepreferably selected from the group consisting of tumor cells and virallyinfected cells. In one suitable instance, the natural or modified enzymeis a non-human PNP or hydrolase. More preferably, the hydrolase is anucleoside hydrolase. Alternatively, the enzyme is a modified mammalianPNP or hydrolase. PNP includes subgroups such as the MTAP(methylthioadenosine phosphorylase).

[0035] In one embodiment of this method of the present invention, theenzyme is provided by targeting the enzyme to the cells. Morepreferably, the enzyme is targeted to the cells by conjugating theenzyme to an antibody.

[0036] The enzyme may be encoded by a gene provided to the cells. Forexample, the gene provided to the cells encodes E. coli PNP and isoperably linked to a tyrosinase gene promoter. Alternatively, the geneis provided in a carrier molecule such as polymeric films, gels,microparticles and liposomes.

[0037] In another embodiment, the present invention provides a method ofkilling replicating or non-replicating, targeted mammalian cells andbystander cells, comprising the following steps: (a) delivering a purinenucleoside phosphorylase to the targeted mammalian cells; and (b)contacting the targeted cells with an effective amount of a nucleosidesubstrate for the purine nucleoside phosphorylase, wherein the substrateis non-toxic to mammalian cells and is cleaved by the phosphorylase toyield a purine base which is toxic to the targeted mammalian cells andbystander cells, to kill the mammalian cells expressing thephosphorylase and the bystander cells. Representative examples of purineanalog substrates include9-(β-D-2-deoxyerythropentofuranosyl)-6-methypurine, 2-amino-6-chloro- 1-deazapurine riboside, 7-ribosyl-3 -deazaguanine,arabinofuranosyl-2-fluoroadenine, 2-fluoro-20-deoxyadenosine,2-fluoro-50-deoxyadenosine, 2-chloro-20-deoxyadenosine,50-amino-50-deoxy-adenosine, α-adenosine, MeP-20,30-dideoxyriboside,2-F-20,30-dideoxyadenosine, MeP-30-deoxyriboside, 2-F-30-deoxyadenosine,2-F-adenine-60-deoxy-β-D-allofuranoside, 2-F-adenine-α-L-lyxofuranoside,MeP-60-deoxy-β-D-allofuranoside, MeP-α-L-lyxofuranoside,2-F-adenine-60-deoxy-α-L-talofuranoside,MeP-60-deoxy-α-L-talofuranoside.

[0038] The present invention also provides a composition for killingtargeted mammalian cells, comprising: (a) an enzyme that cleaves apurine substrate; and (b) an effective amount of the purine analogsubstrate to kill the targeted cells when cleaved by the enzyme.

[0039] The present invention is also directed to a vector comprising aDNA sequence coding for a purine nucleoside phosphorylase protein andsaid vector is capable of replication in a host which comprises, inoperable linkage: a) an origin of replication; b) a promoter; and c) aDNA sequence coding for said protein. Preferably, the vector is selectedfrom the group consisting of a retroviral vector, an adenoviral vector,an adeno-associated viral vector, a herpes vector, a viral vector and aplasmid.

[0040] The present invention also includes a method for inhibiting tumorgrowth by directly administering to a tumor a purine analog orderivative thereof.

[0041] The present invention is also directed to a host cell transfectedwith the vector of the present invention so that the vector expresses anE. coli purine nucleoside phosphorylase protein. Preferably, such hostcells are selected from the group consisting of bacterial cells,mammalian cells and insect cells.

[0042] Some of the methods and compositions, exemplified below, involvetransfecting cells with the E. coli DeoD gene (encoding a purine analognucleoside phosphorylase (PNP)) and subsequently treating with anontoxic purine nucleoside, e.g., deoxyadenosine or deoxyguanosineanalogs, including N7 analogs), which is converted to a toxic purineanalog. E. coli PNP differs from human PNP in its more efficientacceptance of adenine and certain guanine-containing nucleoside analogsas substrates. E. coli PNP expressed in tumor cells cleaves thenucleoside, liberating a toxic purine analog. Purine analogs freelydiffuse across cell membranes, whereas nucleoside monophosphates such asthose generated using HSV Thd kinase, generally remain inside the cellin which they are formed. A toxic adenine analog formed after conversionby E. coli PNP can be converted by adenine phosphoribosyl transferase totoxic nucleotides and kill all transfected cells, and diffuse out of thecell and kill surrounding cells that were not transfected (bystandercells).

Enzymes Catalyzing Purine Analog Conversion

[0043] Two classes of enzymes can be used: phosphorylases andnucleosidase hydrolases. A PNP useful in the methods and compositionsdescribed herein catalyzes the conversion of purine analog nucleosidesplus inorganic phosphate to free the toxic purine analog plus ribose-1-phosphate (or deoxyribose-1-phosphate): purine analog nucleoside+PO₄:purine analog ribose-1 -PO₄ (or deoxyribose- 1-phosphate)+toxic purineanalog. Methylthioadenosine phosphorylase, a subclass of PNP, would alsobe useful in this context. Non-mammalian and modified human or modifiedother mammalian PNPs can be used. The non-mammalian PNP can be an E.coli purine analog nucleoside phosphorylase. However, any PNP which canselectively convert a substrate to produce a toxic purine analog can beutilized. Thus, modifications in the E. coli PNP, which retain thisactivity, are within the scope of the class of enzymes suitable for thedescribed methods and compositions, as are human PNP enzyme moleculesthat have been modified to cleave purine analog nucleoside to releasethe toxic purine analog moiety. A method is presented below by which anyproposed PNP or other purine analog nucleoside cleavage enzyme can betested in a cell for its ability to convert a given substrate from arelatively nontoxic form to a toxin for the cells.

[0044] Table I lists organisms which possess an enzyme that cleavesadenine-containing nucleosides to adenine and so are useful in themethods described herein. Table I also shows that humans and the malariaparasite Plasmodium falciparum do not possess an enzyme useful in thedescribed methods. Thus, to be useful in the methods described herein, ahuman or P. falciparum PNP would have to be modified torbe capable ofcleaving a purine analog nucleoside substrate to liberate this toxicpurine analog. Such modifications can be made at the genetic level orprotein level. For example, in vitro mutagenesis of the gene encodingthe human or P. falciparum PNP can be used to alter the gene sequence toencode a PNP that will cleave a particular purine analog nucleoside.

[0045] As described above, in a preferred embodiment, the PNP used inthe present methods can include genetically modified mammalian ornon-mammalian PNP, as well as bacterial PNP, capable of reacting with asubstrate that the native PNP in the tumor cell will not recognize orrecognizes poorly. Thus, the nucleic acids that encode a useful PNP arepresent in cells in which they are not naturally found, either becausethey are from a different organism or because they have been modifiedfrom their natural state. The key requirement of the nucleic acidsencoding the PNP or other purine analog nucleoside cleavage enzyme isthat they must encode a functional enzyme that is able to recognize andact upon a substrate that is not well recognized by the native PNP ofthe cell.

[0046] Nucleosidases or nucleoside hydrolases are another class ofenzymes suitable for the methods and compositions described herein. Thedefinition of a purine analog nucleosidase is an enzyme that catalyzesthe conversion of purine analog nucleosides plus water to liberate freetoxic purine analogs plus ribose (or deoxyribose): purine analognucleoside+H₂O: purine analog+ribose (or deoxyribose).

Transcriptional Regulation of the PNP Encoding Sequence

[0047] Since a bacterial PNP is encoded on a prokaryotic gene, theexpression of the bacterial PNP in mammalian cells will require aeukaryotic transcriptional regulatory sequence linked to thePNP-encoding sequences. The bacterial PNP gene can be expressed underthe control of strong constitutive promoter/enhancer elements that areobtained within commercial plasmids (for example, the SV40 earlypromoter/enhancer (pSVK30 Pharmacia, Piscataway, N.J., cat. no.27-4511-01), moloney murine sarcoma virus long terminal repeat (pBPV,Pharmacia, cat. no. 4724390-01), mouse mammary tumor virus long terminalrepeat (pMSG, Pharmacia, cat. no. 27-4506-01), and the cytomegalovirusearly promoter/enhancer (pCMVβ, Clontech, Palo Alto, Calif., cat. no.6177-1)).

[0048] Selected populations of cells can also be targeted fordestruction by using genetic transcription regulatory sequences thatrestrict expression of the bacterial PNP (or other suitable purineanalog nucleoside cleavage enzyme) coding sequence to certain celltypes, a strategy that is referred to as “transcription targeting. ” Acandidate regulatory sequence for transcription targeting must fulfilltwo important criteria as established by experimentation: (i) theregulatory sequence must direct enough gene expression to result in theproduction of enzyme in therapeutic amounts in targeted cells, and (ii)the regulatory sequence must not direct the production of sufficientamounts of enzyme in non-targeted cells to impair the therapeuticapproach. In this form of targeting, the regulatory sequences arefunctionally linked with the PNP sequences to produce a gene that willonly be activated in those cells that express the gene from which theregulatory sequences were derived. Regulatory sequences that have beenshown to fulfill the criteria for transcription targeting in genetherapy include regulatory sequences from the secretory leucoproteaseinhibitor, surfactant protein A, and α-fetoprotein genes. A variation onthis strategy is to utilize regulatory sequences that confer“inducibility” so that local administration of the inducer leads tolocal gene expression. As one example of this strategy,radiation-induced sequences have been described and advocated for genetherapy applications. It is expected that bacterial PNP gene expressioncould be targeted to specific sites by other inducible regulatoryelements.

[0049] It may be necessary to utilize tissue-specific enhancer/promotersas a means of directing PNP expression, and thereby PNP-mediatedtoxicity, to specific tissues. For example, human tyrosinase geneticregulatory sequences are sufficient to direct PNP toxicity to malignantmelanoma cells. Mouse tyrosinase sequences from the 50 flanking region(−769 bp from the transcriptional start site) of the gene were capableof directing reporter gene expression to malignant melanoma cells.Although the mouse and human tyrosinase sequences in the 50 flankingregion are similar, Shibata et al., Journal of Biological Chemistry,267:20584-20588 (1992) have shown that the human 50 flanking sequencesin the same region used by Vile and Hart (−616 bp from thetranscriptional start site) did not confer tissue specific expression.Although Shibata et al. suggested that the 50 flanking region would notbe useful to target gene expression to tyrosinase expressing cells(melanomas or melanocytes), a slightly different upstream fragment fromthat used by Shibata et al., can in fact direct reporter or bacterialPNP gene expression specifically to melanoma cells, as shown in FIG. 3.

[0050] The 50 flanking region of the human tyrosinase gene was amplifiedby the polymerase chain reaction from human genomic DNA. The primerswere designed to amplify a 529 bp fragment that extended −451 to +78 bprelative to the transcription start site by using a published sequenceof the human tyrosinase gene and flanks (Kikuchi, et al., Biochimica etBiophysica Acta, 1009:283-286 (1989)). The fragment was shown byreporter gene assays to be able to direct reporter gene expression inmelanoma cells (FIG. 2). The same tyrosinase fragment was used to directPNP expression within a plasmid vector and shown to result in PNPmediated toxicity only in melanoma cells (FIG. 3). Therefore, humantyrosinase sequences are useful to direct PNP expression to humanmelanoma cells. These same sequences could be useful to direct othertherapeutic gene expression in melanoma cells or melanocytes. Othertissue-specific genetic regulatory sequences and elements can be used todirect expression of a gene encoding a suitable purine analog nucleosidecleavage enzyme to specific cell types other than melanomas. TABLE IOrganism Enzyme Organisms which can cleave adenine-containingnucleosides to adenine Leishmania donvani Hydrolase Trichomomasvaginalis Phosphorylase Trypanosoma cruzi Hydrolase Schistosoma mansoniPhosphorylase Leishmania tropica Hydrolase Crithidia FasciculataHydrolase Aspergilis and Penicillium Hydrolase Erwinia carotovoraPhosphorylase Helix pomatia Phosphorylase Ophiodon elongatus (lingcod)Phosphorylase E. coli Phosphorylase Salmonella typhimurium PhosphorylaseBacillus subtilis Phosphorylase Clostridium Phosphorylase mycoplasmaPhosphorylase Trypanosoma gambiense Hydrolase Trypanosoma bruceiPhosphorylase (methylthio adenosine phosphorylase) Organisms whichcannot (or poorly) convert adenine-containing nucleosides to adenineHuman Phosphorylase P. falciparum Phosphorylase

Substrate Selection

[0051] A purine analog nucleoside which is a substrate for the enzyme toproduce a toxic substance which kills the cells is referred to herein asa “prodrug.” Any deoxypurine analog nucleoside composed of the cytotoxicpurine bases including those listed below and in Table II should be asubstrate for the E. coli PNP or other equivalent purine analognucleoside cleavage enzyme. A requisite is that the analog must have alow toxicity at the nucleoside level (that is, as a prodrug). Usingribose- or deoxyribose-containing substrates, E. col PNP can selectivelyproduce a variety of toxic guanine analogs, such as 6-thioguanine or3-deazaguanine, that are attached to ribose or deoxyribose via the N-7position in the guanine ring. The strategy described here fortherapeutic PNP gene transfer implicates new uses for several broadclasses of specifically activatable cytotoxic purine analogs in thetreatment of human malignancy. Because the growth fraction is very smallin most tumors, it is sometimes preferable to select compounds that areactive against both dividing and nondividing cells. Some of the toxicpurine analogs produced using E. coli PNP in the present method aretoxic to nondividing as well as dividing cells. Specific examples ofsuitable purine analog nucleosides that will work in the compositionsand methods described herein can be tested according to the protocolsset forth in the Examples.

[0052] In a preferred embodiment described in the Examples, thesubstrate is 9-(β-D-2-deoxyerythropentofuranosyl)-6-methylpurine(MeP-dR). Although MeP-dR is relatively non-toxic, the therapeutic indexof this compound can be enhanced. For instance, if the toxicity ofMeP-dR is due to phosphorylation by a deoxynucleoside kinase, thenanalogs that cannot be phosphorylated, such as 50-deoxy-MeP-dR, can besynthesized and used as the prodrug to generate MeP in vivo.

[0053] The compounds 6-methylpurine-20-deoxyriboside (Gene Therapy,1:233-238, 1994), 2-amino-6-chloro-1-deazapurine riboside (Biochem.Pharmacol., 33:261-271, 1984), and 7-ribosyl-3-deazaguanine (Biochem.Pharmacol., 29:1791 - 1787, 1979) are examples of prodrugs that areuseful substrates for the E. coli PNP. They are much less toxic thantheir respective purine analogs.

Delivery of the PNP Gene

[0054] Described below is the construction of suitable recombinantviruses and the use of adenovirus for the transfer of bacterial PNP intomammalian cells. Non-viral gene delivery can also be used. Examplesinclude diffusion of DNA in the absence of any carriers or stabilizers(“naked DNA”), DNA in the presence of pharmacologic stabilizers orcarriers (“formulated DNA”), DNA complexed to proteins that facilitateentry into the cell (“Molecular conjugates”), or DNA complexed tolipids. The use of lipid-mediated delivery of the bacterial PNP gene tomammalian cells is exemplified below. More particularly, cationicliposome-mediated transfer of a plasmid containing a non-human PNP geneis demonstrated. However, other gene transfer methods will alsogenerally be applicable because the particular method for transferringthe PNP gene to a cell is not solely determinative of successful tumorcell killing. Thus, gene transduction, utilizing a virus-derivedtransfer vector, further described below, can also be used. Such methodsare well known and readily adaptable for use in the gene-mediated toxintherapies described herein. Further, these methods can be used to targetcertain diseases and cell populations by using the targetingcharacteristics of a particular carrier of the gene encoding a suitablepurine analog nucleoside cleavage enzyme such as E. coli PNP.

[0055] Apathogenic anaerobic bacteria have been used to selectivelydeliver foreign genes into tumor cells. For example, Clostridiumacetobutylicum spores injected intravenously into mice bearing tumors,germinated only in the necrotic areas of tumors that had low oxygentension. Using the standard PNP assay described below, Clostridiumperfringens (Sigma Chemical Co., St. Louis, Mo.) was found to exhibitenzyme activity capable of converting MeP-dR to MeP. This findingsuggests a mechanism to selectively express bacterial PNP activity intumor masses with necrotic, anaerobic centers. Thus, tumors can beinfected with such strains of Clostridium and then exposed to a purineanalog such as MeP-dR. The PNP activity of the clostridium bacteriagrowing in the anaerobic center of the tumor tissue should then convertthe MeP-dR to MeP, which then is released locally to kill the-tumorcells. Additionally, other bacteria including E. coli and Salmonella canbe used to deliver the PNP gene or hydrolase into tumors. As describedand demonstrated below in Example 25, E. coli containing a plasmid (seeFIG. 12) encoding the E. coli PNP gene plus MeP-dR demonstratedefficacious anti-tumor activity (slowing of tumor growth) in mice andalso that delivery of significant amounts of E. coli PNP to tumor cellsin animals could activate MeP-dR resulting in anti-tumor response.

[0056] The rapidly advancing field of therapeutic DNA delivery and DNAtargeting also includes vehicles such as “stealth” and otherantibody-conjugated liposomes (including lipid-mediated drug targetingto colonic carcinoma), receptor-mediated targeting of DNA through cellspecific ligands, lymphocyte-directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells in vivo(S. K. Huang et al., Cancer Research, 52:6774-6781 (1992); R. J. Debs etal., Am. Rev. Respir. Dis., 135:731-737 (1987); K. Maruyama et al.,Proc. Natl. Acad. Sci. USA, 87:5744-5748 (1990); P. Pinnaduwage and L.Huang, Biochemistry, 31:2850-2855 (1992); A. Gabizon and et al., NewEngland J. Med., 323:570-578 (1990); K. Culver et al., Proc. Natl. Acad.Sci. USA, 88:3155-3159 (1991); G. Y. Wu and C. H. Wu, J. Biol. Chem.,263, No. 29:14621-14624 (1988); Wagner et al., Proc. Natl. Acad. Sci.USA, 87:3410-3414 (1990); Curiel et al., Human Gene Ther., 3:147-154(1992); Litzinger, Biochimica et Biophysica Acta, 1104:179-187 (1992);Trubetskoy et al., Biochemica et Biophysica Acta, 1131:311-313 (1992)).The present approach, within the context of a gene targeting mechanismeither directed toward dividing tumor cells or tumor neovascularization,offers an improved means by which a small subset of tumor cells could beestablished within a growing tumor mass, which would mediate rapid tumorinvolution and necrosis after the appropriate signal, i.e., afteradministration of the substrate (prodrug) for a suitable purine analognucleoside cleavage enzyme, such as E. coli PNP present in or adsorbedto tumor cells.

Methods of Treatment

[0057] The method of treatment basically consists of providing to cellsthe PNP gene and then exposing the cells with the PNP gene or protein toan appropriate substrate which is converted to a toxic substance whichkills the cells expressing the PNP gene as well as those in the vicinityof the PNP gene expressing cells. The PNP gene can be administereddirectly to the targeted cells or systemically in combination with atargeting means, such as through the selection of a particular viralvector or delivery formulation. Cells can be treated in vivo, within thepatient to be treated, or treated in vitro, then injected into thepatient. Following introduction of the PNP gene into cells in thepatient, the prodrug is administered, systemically or locally, in aneffective amount to be converted by the PNP into a sufficient amount oftoxic substance to kill the targeted cells.

Treatment of Tumors

[0058] The E. coli PNP gene can also be used as part of a strategy totreat metastatic solid tumors, such as melanoma, pancreatic, liver orcolonic carcinoma. No effective therapy for metastatic tumors of thesetypes currently exists. In this method, plasmid DNA containing a PNPgene under the control of tumor specific promoters is used. For example,the tyrosinase promoter is highly specific for mediating expression inmelanoma cells, and will not lead to gene expression in most tissuetypes. The PNP gene under the regulatory control of this promoter,therefore, should be activated predominantly within a melanoma tumor andnot elsewhere within a patient (see Example 11 and FIGS. 2A-D below).Promoters specific for other tumor types, for example, promoters activein the rapidly dividing endothelial cells present in all solid tumorscan be used to specifically activate PNP only within a primary ormetastatic tumor. In this method, plasmid DNA containing PNP under thecontrol of a tumor specific promoter is delivered to cells usingcationic liposomes. For example, based on animal studies, 100-400 mgplasmid DNA complexed to 1200-3600 micromoles of a 1:1 mixture of thelipids DOTMA (1,2-dioleyloxypropyhl-3-trimethyl ammonium bromide) andDOPE (dioleoyl phosphatidylethanolamine) could be used to deliver thePNP gene to tumor metastases in patients. A prodrug in the abovedescribed amounts can then be administered.

[0059] The PNP gene can be used to activate prodrugs in the treatment ofhuman brain cancer. In this method, a cell line producing retroviralparticles, in which the viral particles contain the E. coli PNP gene, isinjected into a central nervous system (CNS) tumor within a patient. AnMRI scanner is used to appropriately inject the retroviral producer cellline to within the tumor mass. Because the retrovirus is fully activeonly within dividing cells and most of the dividing cells within thecranium of a cancer patient are within the tumor, the retrovirus isprimarily active in the tumor itself, rather than in non-malignant cellswithin the brain. Clinical features of the patient including tumor sizeand localization, determine the amount of producer cells to be injected.For example, a volume of producer cells in the range of 30 injections of100 microliters each (total volume 3 ml with approximately 1×10⁸producer cells/ml injected) are given under stereotactic guidance forsurgically inaccessible tumors. For tumors which can be approachedintraoperatively, 100 μl aliquots are again injected (at about 1×10⁸cells/ml) with total injected volumes up to 10 ml using E. coli PNP genetransfer, followed by MeP-dR administration. This strategy is designedto permit both bystander killing and toxicity to non-dividing cells andis thus designed for much greater tumor involution than previousattempts using HSV dThd kinase and ganciclovir.

[0060] The destruction of selected populations of cells can be achievedby targeting the delivery of the bacterial PNP gene or other geneencoding an enzyme capable of cleaving purine analog from a purineanalog nucleoside (such as adenine from adenine-containing nucleosidesas described above). The natural tropism or physiology of viral vectorscan also be exploited as a means of targeting specific cell types. Forexample, retroviruses are well known to become fully active only inreplicating cells. This fact has been used as the basis for selectiveretroviral-mediated gene transfer to replicating cancer cells growingwithin a site where the normal (nonmalignant) cells are not replicatingin both animal and human clinical studies. Alternatively, the viralvector can be directly administered to a specific site such as a solidtumor, where the vast majority of the gene transfer will occur relativeto the surrounding tissues. This concept of selective delivery has beendemonstrated in the delivery of genes to tumors in mice by adenovirusvectors. Molecular conjugates can be developed so that the receptorbinding ligand will bind only to selective cell types, as has beendemonstrated for the lectin-mediated targeting of lung cancer.

[0061] Recently, it Was shown that intravenous injection of liposomescarrying DNA can mediate targeted expression of genes in certain celltypes. Targeting of a gene encoding a purine analog nucleoside cleavageenzyme or expression of the gene to a small fraction of the cells in atumor mass followed by substrate administration could be adequate tomediate involution. Through the substantial bystander effect and killingof nondividing cells demonstrated in the Examples, the present methodcan be used to destroy the tumor.

Treatment of Virally Infected Cells

[0062] In addition to killing tumor cells, the methods described hereincan also be used to kill virally infected cells. In a virus-killingembodiment, the selected gene transfer method is chosen for its abilityto target the expression of the cleavage enzyme in virally infectedcells. For example, virally infected cells may utilize special viralgene sequences to regulate and permit gene expression, that is, virusspecific promoters. Such sequences are not present in uninfected cells.If the PNP gene is oriented appropriately with regard to such a viralpromoter, the cleavage enzyme would only be expressed within virallyinfected cells, and not other, uninfected, cells. In this case, virallyinfected cells would be much more susceptible to the administration ofMeP-dR or other substrates designed to be converted to toxic form bynon-human or modified human purine nucleoside cleavage enzyme.

Administration of Genetically Engineered Cells

[0063] For certain applications, cells that receive the PNP gene areselected and administered to a patient. This method most commonlyinvolves ex vivo co-transfer of both the gene encoding the cleavageenzyme, such as the bacterial PNP gene, and a second gene encoding atherapeutic protein gene. The cells that receive both genes arereinfused into the host patient where they can produce the therapeuticprotein until the prodrug, such as MeP-dR, is administered to eliminatethe engineered cells. This method should be useful in “cell therapies”,such as those used on non-replicating myoblasts engineered for theproduction of tyrosine hydroxylase within the brain (Jiao et al.,Nature, 362:450 (1993)).

Direct Delivery of the PNP Enzyme to Cells

[0064] The bystander killing conferred by the bacterial PNP protein plusprodrug combination can also be achieved by delivering the PNP proteinto the target cells, rather than the PNP gene. For example, a PNP enzymecapable of cleaving purine analog nucleosides as described above, ismanufactured by available recombinant protein techniques usingcommercially available reagents. As one example of a method forproducing the bacterial PNP protein, the E. coli PNP coding sequence isligated into the multiple cloning site of pGEX-4T-1 (Pharmacia,Piscataway N.J.) so as to be “in frame”, with theglutathione-s-transferase (GST) fusion protein using standard techniques(note that the cloning site of this vector allows insertion of codingsequences in all three possible translational reading frames tofacilitate this step). The resulting plasmid contains the GST-PNP fusioncoding sequence under transcriptional control of the IPTG-inducibleprokaryotic tac promoter. E. coli cells are transformed with therecombinant plasmid and the tac promoter induced with IPTG. IPTG-inducedcells are lysed, and the GST-PNP fusion protein purified by affinitychromatography on a glutathione Sepharose 4B column. The GST-PNP fusionprotein is eluted, and the GST portion of the molecule removed bythrombin cleavage. All of these techniques and reagents are provided ina commercially available kit (Pharmacia, Piscataway, N.J., catalog no.27-457001). Other methods for recombinant protein production aredescribed in detail in published laboratory manuals. Since the bacterialPNP activates the prodrugs into diffusible toxins, it is only necessaryto deliver the PNP protein to the exterior of the target cells prior toprodrug administration. The PNP protein can be delivered to targets by awide variety of techniques. One example would be the direct applicationof the protein with or without a carrier to a target tissue by directapplication, as might be done by directly injecting a tumor mass withinan accessible site. Another example would be the attachment of the PNPprotein to a monoclonal antibody that recognizes an antigen on the tumorsite. Methods for attaching functional proteins to monoclonal antibodieshave been previously described. The PNP conjugated monoclonal antibodyis systemically administered, for example, intravenously (IV), andattaches specifically to the target tissue. Subsequent systemicadministration of the prodrug will result in the local production ofdiffusible toxin in the vicinity of the tumor site. A number of studieshave demonstrated the use of this technology to target specific proteinsto tumor tissue. Other ligands, in addition to monoclonal antibodies,can be selected for their specificity for a target cell and testedaccording to the methods taught herein.

[0065] Another example of protein delivery to specific targets is thatachieved with liposomes. Methods for producing liposomes are describede.g., Liposomes: A Practical Approach). Liposomes can be targeted tospecific sites by the inclusion of specific ligands or antibodies intheir exterior surface, in which specific liver cell populations weretargeted by the inclusion of asialofetuin in the liposomal surface (VanBerkel et al., Targeted Diagnosis and Therapy, 5:225-249 (1991)).Specific liposomal formulations can also achieve targeted delivery, asbest exemplified by the so-called StealthJ liposomes that preferentiallydeliver drugs to implanted tumors (Allen, Liposomes in the Therapy ofInfectious Diseases and Cancer, 405-415 (1989)). After the liposomeshave been injected or implanted, unbound liposome is allowed to becleared from the blood, and the patient is treated with the purineanalog nucleoside prodrug, such as MeP-dR, which is cleaved to MeP bythe E. coli PNP or other suitable cleavage enzyme at the targeted site.Again, this procedure requires only the availability of an appropriatetargeting vehicle. In a broader sense, the strategy of targeting can beextended to specific delivery of the prodrug following either PNPprotein, or gene delivery.

Administration of Substrates

[0066] The formula of Freireich et al., Cancer Chemother. Rep.,50:219-244, (1966) can be used to determine the maximum tolerated doseof substrate for a human subject. For example, based on systemicallyadministered dose response data in mice showing that a dose of 25 mg(Mep-dR) per kg per day for 9 days (9 doses total) resulted in sometoxicity but no lethality, a human dosage of 75 mg MeP-dR/m² wasdetermined according to the formula: 25 mg/kg×3=75 mg/M². This amount orslightly less should result in maximal effectiveness of tumor cellkilling in humans without killing the subject. This standard ofeffectiveness is accepted in the field of cancer therapy. However, morepreferred is a range of from about 10% to 1% of the maximum tolerateddosage (for example, 7.5 mg/M²-0.75 mg/M²). Furthermore, it isunderstood that modes of administration that permit the substrate toremain localized at or near the site of the tumor will be effective atlower doses than systemically administered substrates.

[0067] The substrate may be administered orally, parenterally (forexample, intravenously), by intramuscular injection, by intraperitonealinjection, or transdermally. The exact amount of substrate required willvary from subject to subject, depending on the age, weight and generalcondition of the subject, the severity of the disease that is beingtreated, the location and size of the tumor, the particular compoundused, its mode of administration, and the like. An appropriate amountmay be determined by one of ordinary skill in the art using only routineexperimentation given the teachings herein. Generally, dosage willpreferably be in the range of about 0.5-50 mg/m², when consideringMeP-dR for example, or a functional equivalent.

[0068] Depending on the intended mode of administration, the substratecan be in pharmaceutical compositions in the form of solid, semi-solidor liquid dosage forms, such as, for example, tablets, suppositories,pills, capsules, powders, liquids, or suspensions, preferably in unitdosage form suitable for single administration of a precise dosage. Thecompositions will include an effective amount of the selected substratein combination with a pharmaceutically acceptable carrier and, inaddition, may include other medicinal agents, pharmaceutical agents,carriers, or diluents. By “pharmaceutically acceptable” is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected substrate withoutcausing significant undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

[0069] For solid compositions, conventional nontoxic solid carriersinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose,sucrose and magnesium carbonate. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving or dispersingan active compound with optimal pharmaceutical adjuvants in anexcipient, such as water, saline, aqueous dextrose, glycerol, orethanol, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, for example, sodium acetate ortriethanolamine oleate. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences.

[0070] For oral administration, fine powders or granules may containdiluting, dispersing, and/or surface active agents, and may be presentedin water or in a syrup, in capsules or sachets in the dry state or in anonaqueous solution or suspension wherein suspending agents may beincluded, in tablets wherein binders and lubricants may be included, orin a suspension in water or a syrup. Where desirable or necessary,flavoring, preserving, suspending, thickening, or emulsifying agents maybe included. Tablets and granules are preferred oral administrationforms, and these may be coated.

[0071] Parenteral administration is generally by injection. Injectablescan be prepared in conventional forms, either liquid solutions orsuspensions, solid forms suitable for solution or prior to injection, oras suspension in liquid prior to injection or as emulsions.

Cells Expressing E. coli PNP Substrate

[0072] The effect of MeP-dR on human colon carcinoma cells expressing E.coli PNP substrate was demonstrated. MeP-dR was chosen because it is20-fold less toxic than 6-methylpurine (MeP) to HEp-2 cells and it hasbeen used to detect cultures infected with mycoplasma, becausemycoplasma express an enzyme similar in function to E. coli PNP.

[0073] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1 Cell Lines

[0074] T-84 colon carcinoma cells were grown in Dulbecco's modifiedEagle medium containing F12 nutrient medium (DMEM/F12) (GIBCO/BRL,Gaithersburg, Md.) in 6 well trays to a density of approximately 1-2×10³cells/well (−20% confluency).

EXAMPLE 2 Toxicity of MeP and MeP-dR Within Colon Carcinoma Cells

[0075] Untransfected T-84 colon carcinoma cells were treated withincreasing concentrations of either MeP-dR or MeP. After 5 days thecells were removed from each well and the number of dye excluding cellswere determined with the aid of a hemacytometer. Cells were studied bothat passage 48 (P. 48) and passage 61 (p. 61). MeP was obtained fromSigma Chemical Company (St. Louis, Mo.). MeP-dR was synthesized bystandard methods as described (J. A. Montgomery and K. Howson, J. Med.Chem., 11:48-52 (1968)). The nucleoside and base were dissolved in serumfree DMEM/F12 at a concentration of 1 mg/ml and added directly to 1 mlDMEM/F12 with 10% fetal bovine serum at the concentrations describedbelow in order to cover 1-2×10⁵ cells/well.

[0076] Initial cytopathic effects due to MeP were observed within 24hours (for example, rounding of cells, with some cells detaching fromplate). Viable cells were counted 5 days following addition of drug. Thehigher concentrations (3.75 μM-75 μM) of MeP resulted in cell lysis andcomplete loss of cellular architecture, leaving only cellular debriswithin wells by day 2 following treatment. Trypan blue exclusion wasused to confirm viability in cells retaining recognizable structure atall concentrations studied. At lower concentrations MeP-dR did not causeany appreciable cell death and higher concentrations (200 and 400 μM)less than half of the cells were killed. If the toxicity of MeP-dR isdue to very low levels of liberation of MeP by human PNP, thencombination with selective inhibitors of human PNP could prevent thistoxicity.

[0077] The relative toxicity of the prodrug, MeP-dR, and the product,MeP, on wild type melanoma cell viability, was tested. Mel-1 cells wereincubated in various concentrations of MeP-dR and MeP for five days. TheMel-1 cells were unaffected by concentrations of MeP-dR as high as 50μg/ml while concentrations of the MeP as low as 0.5 μg/ml were nearly100% lethal. Similar results have been obtained in T-84, B16, and 16/Ccells. Both MeP-dR and MeP are stable under tissue culture conditions asmeasured by HPLC analysis of supernatants.

EXAMPLE 3 Synthesis of E. coli PNP Expression Vectors

[0078] A bacterial PNP-encoding sequence was inserted into a plasmidexpression vector. E. coli (strain, JM101) chromosomal DNA template wasobtained using the method described in N. J. Gay, J. Bacteriol.,158:820-825 (1984). Two PCR primersGATCGCGGCCGCATGGCTACCCCACACATTAATGCAG (SEQ ID NO: 1) andGTACGCGGCCGCTTACTCTTTATCGCCCAGCAGAACGGA-TTCCAG (SEQ ID NO: 2) were usedto define the full length coding sequence of the E. coli DeoD gene andto incorporate Notl sites at both 50 and 30 ends of the desired product.After 30 cycles of amplification (94° C.×1 minute denaturation, 50° C.×2minute annealing, and 72° C.×3 minute elongation using 1 ng template,100 μl of each primer in a 100 μl reaction mixture containing 2.5 unitstaq polymerase, 200 μM each dNTP, 50 mM KCl, 10 mM Tris Cl (pH 8.3), 1.5mM MgCl₂ and 0.01% gelatin (weight/vol)), a single PCR product of thepredicted size (716 base pairs) was obtained. This product was extractedwith phenol/chloroform, precipitated with ethanol, digested with NotI,and gel purified using the Gene clean kit (Bio. 101, La Jolla, Calif.).

[0079] The amplified bacterial PNP sequence was added to a plasmideukaryotic expression vector. In order to obtain a vector capable ofdirecting eukaryotic expression of E. coli PNP, the LacZ gene wasexcised from pSVB (Clontech, Palo Alto, Calif.) by digestion with NotI,the vector backbone was dephosphorylated (calf intestinal alkalinephosphatase, GIBCO BRL, Gaithersburg, Md.) and gel purified as above.The PNP insert, prepared as above, was then ligated into the Notl endsof the plasmid backbone in order to create a new construct with PNPexpression controlled by the SV-40 early promoter. Correct recombinants(and orientation of inserts) were confirmed by restriction mapping(using twelve restriction digests which cut in both vector and insert),and by reamplification of the full length insert from recombinantplasmid using the primers described above. This procedure yielded theplasmid SV-PNP.

EXAMPLE 4 Transfection of T-84 Colon Carcinoma Cells

[0080] Cationic liposome mediated gene transfer was used to transfectT-84 colon carcinoma cells. Briefly, 6 μg of plasmid containing PNP orLacZ was added to 10 μg of a 1:1 molar mixture of DOTMA/DOPE(LipofectinJ (GIBCO/BRL, Gaithersburg, Md.)) in a final volume of 200 μlDMEM/FI2 serum free medium. After a 10 minute incubation at roomtemperature, the DNA-lipid mixture was added to 500 μl serum free mediumand was used to cover the cells within a tray. Four hours later,transfection medium was removed from each well and 2 ml DMEM/FI2 with10% fetal bovine serum was added.

EXAMPLE 5 Transfection Efficiency

[0081] The LacZ gene was transfected into T-84 cells as described above.Briefly, using a lipid-mediated gene transfer protocol identical to thatdescribed above, 6 μg of plasmid containing the E. coli LacZ gene underthe control of the SV-40 early promoter was transferred into 1-2×10⁵T-84 cells. 48 hours after transfection, cells were washed 3 times inPBS, fixed at 4° C.×10 minutes in 0.2% glutaraldehyde, (in 80 mMNaHPO₂), rinsed 2 times with PBS, and then stained in a solutioncontaining 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 1.3 mM MgCl₂, 3 mM K₃Fe(CN)₆, 3mM K₄Fe(CN)₆ and 1 mg/ml x-gal(5-bromo-4-chloro-3-indolyl-β-D-galactosidase). 12 hours after staining,0.1-1% of the cells treated with β-galactosidase DNA stained positivefor gene expression.

[0082] X-gal staining of these cells two days after transfectionindicated an overall transfection efficiency of 0.1-1% (as determined bypercentage of blue cells). No positive cells were observed in untreatedT-84 cells, in cells treated with lipid alone, or plasmid DNA alone.Similar conclusions were reached using a LacZ reporter gene containing anuclear targeting sequence and leading to nuclear staining ofrecombinant cells.

EXAMPLE 6 Toxicity of MeP-dR-mediated by E. coli PNP Expression Vectors

[0083] Forty-eight hours following transfection, fresh medium was addedand MeP-dR (1 mg/ml in PBS) was added directly to the cells to achievethe desired final concentrations. Cell viability was measured 5 daysfollowing treatment as described for the MeP-dR toxicity study.

[0084] In one experiment, MeP-DR (160 μM) was added to wells containinguntransfected cells, or cells transfected with 10, 20, or 40 μg (FIG. 1)of cDNA containing either the E. coli PNP or LacZ genes under control ofthe SV-40 early promoter (in otherwise comparable vector contexts).After 5 days the cells were removed from each well and the number of dyeexcluding cells were determined with the aid of a hemacytometer. 30-50%toxicity due to the DOTMA-DOPE transfection protocol is acceptable forcationic liposome mediated gene transfer to T-84 in vitro when performedunder optimal conditions. The results of this study are shown in FIG. 1.

[0085] In an additional experiment, approximately 2×10⁵ cells per wellwere transfected as above using 6 μg of plasmid containing E. coli PNPcDNA. Two days after transfection, varying concentrations of MeP-dR (0,2, 4, 20, 40 and 160 μM) were added to the wells, and after 5 days thedye excluding cells were counted with the aid of a hemacytometer.Concentrations of MeP-dR as low as 4 μM resulted in greater than 80%inhibition of cell growth. An experiment was also performed intriplicate in which 2×10⁵ cells per well were transfected with LacZ orPNP using the protocol described for transfection. Two days aftertransfection, 16 μM MeP-dR was added to one set of the culturestransfected with PNP and one set of the cultures transfected with LacZ.The other PNP and LacZ transfected cultures did not receive drug. Theresults demonstrate minimal cell killing in all cultures except the PNPtransfected, MeP-dR treated culture.

[0086] In the above experiments, MeP-dR (160 μM) was minimally toxic tothe cells that were not transfected. While expression of the LacZ genehad no influence on toxicity mediated by MeP-dR, MeP-dR killed virtuallyall of the cells transfected with the E. coli PNP (FIG. 1). Substantialkilling could also be seen with 16 μM MeP-dR after PNP transfection.These results indicate that low efficiency expression of E. coli PNPcDNA (expression in less than 1% of tumor cells) was adequate for nearly100% transfected cell and bystander cell killing. In addition, becausediffusion of MeP into the medium covering the cells could have asubstantial dilutional affect, it may be that an even lower fraction oftumor cells expressing E. coli PNP in vivo might be able to mediatetumor cell necrosis in the presence of MeP-dR.

EXAMPLE 7 Activity of E. coli PNP on MeP-dR in Cell Extracts

[0087] The toxicity of MeP-dR in T-84 cells expressing the E. coli PNPactivity was measured in transfected T-84 cells. Briefly, T-84 cellstransfected with 6 μg of plasmid containing either the E. coli PNP geneor the LacZ (β-galactosidase) gene as described above were collected bycentrifugation 48 hours after transfection and resuspended in 3 volumesof 0.01 M potassium phosphate (pH 7.4), followed by incubation on icefor 15 minutes. The pellet was homogenized, and the sample wascentrifuged at 100,000×g for 60 minutes. PNP activity was measured in100 μl volumes containing 50 mM potassium phosphate (pH. 7.4), 100 μM ofMeP-dR, and 1 mg/ml of protein from the cell extract. After incubationfor 24 hours at 25° C., the reaction was stopped by boiling, theprecipitated proteins were removed by centrifugation, and the reactionmixture was subjected to HPLC by injection onto a Spherisorb ODSI (5 μm)column (Keystone Scientific Inc., State College, Pa.). The MeP-dR andMeP were eluted with a 30 min isocratic gradient of 50 mM ammoniumdihydrogen phosphate buffer (pH 4.5)/acetonitrile (95/5; v/v) at a flowrate of 1 ml/minute. MeP-dR and MeP were detected by their absorbance at254 nm.

[0088] Approximately 24% of the MeP-dR was converted to MeP in extractsfrom the T-84 colon carcinoma cells transfected with the E. coli PNPgene, whereas no conversion occurred in cell extracts from coloncarcinoma cells transfected with the LacZ gene. Total PNP activity(human+E. coli) measured using inosine as substrate was not changed inT-84 cells transfected with E. coli PNP. Thus, despite the relativelylow level of expression of the E. coli PNP in the transfected cells, asufficient amount of the MeP-dR was converted to kill all of the cells.

EXAMPLE 8 Detection of MeP in Medium of T-84 Cells Transfected with E.coli PNP

[0089] MeP-dR (160 μM) was added 48 hours after transfection of T-84cells with the E. coli PNP gene. Five days after the addition of MeP-dR,the medium was collected, and the proteins were precipitated by boiling.After centrifugation, the medium was analyzed for the appearance of MePby reverse phase HPLC as described above.

[0090] MeP was detected only in the culture medium of T-84 cellstransfected with E. coli PNP. More than 75% of the MeP-dR was convertedto MeP over a 5 day period in E. coli PNP transfected cells, but not inLacZ transfected cells. These results have significance, because theyindicated that 1) untransfected and mock transfected colonic carcinomacells lack an enzymatic mechanism for conversion of MeP-dR to MeP, 2) aspredicted, MeP was readily released into the extracellular medium, so asto establish effective bystander killing, and 3) the extracellularconcentrations of MeP generated by recombinant PNP were sufficient tofully explain the bystander killing which was observed. In addition,these results establish that SV-40 driven expression of the prokaryoticPNP in eukaryotic cells (as with the E. coli LacZ) leads to a highlyactive and functional enzyme. Because E. coli PNP is believed toassemble as a homohexamer in prokaryotic cells, the mechanisms of E.coli PNP oligomerization are likely to be compatible with eukaryoticprotein synthesis.

EXAMPLE 9 Toxicity to Nondividing Cells

[0091] Results from experiments indicate that MeP is able to killnon-proliferating cells. This distinguishes MeP from most otherantitumor agents. In the first experiment, CEM cells were cultured in 1%serum instead of the normal 10% serum for 48 hours. Under theseconditions, the cells stop growing and the cell numbers stabilize at 1.5to 2 times the original cell numbers. Cell growth continues when cellsare returned to culture medium containing 10% serum. Addition of MeP ata final concentration of 10 μg/ml to CEM cell cultures after 48 hours ofincubation with 1% serum caused a decline in cell numbers toapproximately 25% of their original number which indicated that MeP wastoxic to non-proliferating cells.

[0092] In the second experiment, the effect of MeP on the incorporationof thymidine into DNA, uridine into RNA, and leucine into protein wasdetermined. RNA and protein synthesis were affected most by treatmentwith MeP. Effects on DNA synthesis occurred only after effects on RNAand protein synthesis were evident. These results indicated that theinhibitory effect of MeP on either RNA or protein synthesis wasresponsible for its toxicity. These two functions are vital to all cellsregardless of their proliferative state, which indicates that MeP shouldbe toxic to both proliferating and non-proliferating cells. Resultsconfirming these conclusions were also obtained in MRC-5 which are anon-transformed human diploid fibroblast cell line derived fromembryonic lung cells.

EXAMPLE 10 Additional Useful Recombinant Vectors

[0093] A recombinant retrovirus was made by adding the bacterial PNPsequences to a plasmid retroviral transfer vector that was subsequentlypassed through packaging cell lines for the production of virus. Theretroviral vector, pLNSX (Miller and Rosman, BioTechniques, 7:980-991(1989)), contains a cloning site that is just 30 to an SV40 earlypromoter which will direct transcription of a coding sequence insertedwithin the cloning site. The bacterial PNP sequence was ligated intolinearized pLNSX. The ligation mixture was used to generate bacterialtransformants that were identified by colony DNA analysis, and one clone(pLN/PNP) containing the PNP coding sequence in a 50 to 30 orientationrelative to the SV40 promoter was amplified by standard techniques andpurified with cesium chloride gradient centrifugation. The plasmid wastransfected by lipid-mediated gene transfer into the ψ2 packaging cellline. The supernatant from these cells was harvested 48 hours later,clarified by 0.45 μM filtration and applied to additional ψ2 packagingcell line. In 24-36 hours, the cells were enzymatically detached andplated at a density ⅕ the original density in media supplemented withG418 (1 g/L). Virus producing cells appeared as colonies 7-10 days laterthat were isolated with cloning rings and assessed for quantity andfidelity of recombinant virus production.

[0094] A recombinant adenovirus was made by adding the bacterial PNPsequences to a plasmid adenoviral vector that was subsequently passedthrough a cell line (293) for the production of virus. The adenoviralplasmid vector, pACCMV (Kolls et al., Proc. Natl. Acad. Sci. USA,91:215-219 (1993)) was linearized with EcoRI and HindIII at the multiplecloning site which is operably linked to the cytomegalovirus (CMV)immediate early promoter. The bacterial PNP-encoding sequence wasexcised from the SV/PNP plasmid using Notl, and the fragment gelpurified and ligated into the NotI site of pSL 1180 (Pharmacia,Piscataway, N.J.) to produce the plasmid designated pSL/PNP. The PNPencoding sequence was excised from pSL/PNP with EcoRI and HindII, gelpurified, and ligated into the EcoRI and HindIII site of pACCMV to makethe new plasmid designated pACCMV/PNP. Transfection of the pACCMV/PNPinto cells conferred dose-dependent toxicity following exposure to theMeP-dR prodrug, confirming that the CMV promoter directed the productionof therapeutic levels of the bacterial PNP. The pACCMV/PNP wascotransduced with the pJMI7 vector into human embryonal carcinoma 293cells that contain adenoviral E1A sequences necessary for viralreplication.

EXAMPLE 11 Tyrosinase Promoter Sequence-directed Expression Plasmids

[0095] The human tyrosinase regulatory sequence was amplified by thepolymerase chain reaction (PCR) from human genomic DNA. The genomic DNAwas obtained from nucleated human blood cells by standard techniques.PCR primers A (GAT CGC TAG CGG GCT CTG AAG ACA ATC TCT CTC TGC (SEQ IDNO. 3)) and B (GAT CGC TAG CTC TTC CTC TAG TCC TCA CAA GGT CT (SEQ IDNO. 4)) amplified bp −451, to +78 with the addition of Nhel restrictionenzyme sites at each end using the sequence of Kikuchio et al., Biochim.Biophys. Acta, 2009:283-286 (1989). The PCR reaction used the followingconditions for 30 cycles: 94° C.×1 min, 50° C.×2 min, 72° C.×3 minutes.The final product was clarified by phenol/chloroform extraction,digested with NheI, gel purified, and ligated into the NheI cloning siteof the commercial luciferase vector, pGL2 Basic (Promega, Madison, Wis.)by standard techniques. Recombinants were screened by restrictionmapping and a correctly oriented clone was identified (Tyr-Luc). Aplasmid with the tyrosinase promoter in reverse orientation(Rev-Tyr-Luc), for use as a negative control, was also selected. Acontrol vector (SV-LUC) containing the SV-40 virus early promoter andSV-40 enhancer region driving the expression of firefly luciferase (pGL2control vector, Promega, Madison, Wis.) was used to verify successfultransfection of cells. To create a plasmid in which the tyrosinasepromoter controlled PNP expression, the PNP gene was substituted forluciferase in the Tyr-Luc. This was accomplished using a XhoI/Sal1digest to excise the full length PNP gene from SV-PNP, followed byinsertion of this fragment into the XhoI/Sal1 sites remaining after aXhoI/Sal1 digest to remove the luciferase gene from Tyr-Luc. Thetyrosinase reporter constructs were tested in transient assays. TheLipofectinJ (GIBCO/BRL, Gaithersburg, Md.) transfection protocol wasused for all luciferase reporter gene experiments. Cells were seeded at50% confluency in six-well plates and allowed to grow overnight.Immediately prior to transfection each well was washed three times withsterile phosphate buffered saline (PBS). A single well of a six-wellplate was transfected with a ratio of 10 μg liposomes/10-20 μg ofplasmid DNA, depending on the cell line. Liposome/DNA complexes wereprepared according to manufacturer's instructions. The liposome/DNAcomplexes were mixed with serum free media (SFM) and a total volume of700 μl was placed in a single well of a six-well plate. After incubationat 37° C. for 14 to 16 hours, the transfection mixture was aspirated and2 ml of complete media was added. The cells were harvested after 48additional hours and luciferase activity was determined using theinstructions and reagents of a commercial kit (Luciferase Assay System,Promega, Madison, Wis.). Luciferase reporter gene expression wasassessed 48, hours following transfection of various carcinoma celllines (melanoma, liver, colon, prostate, myeloma, glial, HeLa) with aconstruct containing a promoterless luciferase vector (“Basic”); aluciferase gene linked to a human tyrosinase promoter in reverseorientation (incorrect orientation to transcribe the luciferase gene)(Rev-Tyr-Luc); a luciferase gene operably linked to the constitutiveSV40 early promoter (SV-Luc); or a luciferase gene operably linked to ahuman tyrosinase promoter (correct orientation to transcribe theluciferase gene) (Tyr-Luc).

[0096] As shown in FIGS. 2A-D, the tyrosinase transcriptional promotersequence specifically restricted expression of the luciferase reportergene to which it was operably linked, to melanoma cells (Mel-1 andMel-21). In contrast, the SV40 early promoter constitutively expressedthe luciferase gene to which it was operably linked in all transfectedcarcinoma cell lines. The results demonstrate that tissue-specificpromoter sequences can be used to transcriptionally target theexpression of a heterologous enzyme to a specific tumor.

[0097] Luciferase activity in Mel-1 and Mel-21 cells transfected withthe Tyr-Luc construct was comparable to luciferase activity generated bytransfection with a plasmid utilizing the SV-40 early promoter tocontrol luciferase gene expression (SV-Luc) (FIG. 2, Panel A). Bothnegative controls (luciferase without promoter (Basic) and luciferasewith tyrosinase promoter sequences inserted in the reverse orientation(Rev-Tyr-Luc)) gave negative results. Negligible Tyr-Luc activity wasseen in five additional human cell lines (T-84-colon cancer, U373-glial,HeLa-cervical carcinoma, RPMI 8226-myeloma, GP6FS-prostate), which allshowed substantial SV-40 driven reporter gene activity (FIG. 2, PanelB-D). In a sixth cell line, Hep G2 (derived from human liver), theSV-Luc was 28 fold more active than the Tyr-Luc. However, the Tyr-Lucvector had activity above background in the Hep G2 cells. Because thepromoterless luciferase vector resulted in similar luciferase activity,luciferase activity in Hep G2 cells is likely to be nonspecific and dueto cryptic promoters or enhancers present within the vector itself,rather than nonspecific regulation by the human tyrosinase promoter.

[0098] To eliminate possible toxicity associated with thenon-hydrolyzable cationic lipid component of the LipofectinJ, analternative liposome transfection vehicle was used in the killingexperiments. A liposome vehicle consisting of a 1:1 (weight/weight)mixture of the cationic lipid DOTAP(1,2-dioleoyloxy-3-(trimethylammonium)-propane) and the neutral lipidDOPE (dioleoyl-phosphatidylethanolamine) (Avanti Polar Lipids) displaytransfection properties similar to LipofectinJ, but with less toxicity(data not shown). DOTAP/DOPE liposomes were prepared by mixing 0.5 mg ofDOTAP and 0.5 mg of DOPE and evaporating the chloroform solvent.Following the addition of 500 μl of cyclohexane, the mixture was placedon dry ice and lyophilized. One ml of sterile water was added to thepowdered lipids and the solution was vortexed every 5 minutes for 30minutes. T-84 or Mel-1 cells were seeded at 30% confluency in 24-wellplates and allowed to grow overnight. Immediately prior to transfection,each well was washed three times with sterile PBS. To transfect a singlewell of a 24-well plate, 7.5 μg of DOTAP/DOPE (1 μg/μl) was mixed with1.875 μg of plasmid DNA (1 μg/μl) and incubated for 15 minutes.Following a 15 minute incubation, the liposome/DNA complexes were mixedwith 266 μl of SFM and added to a single well of a 24-well plate. Theplates were incubated for four hours at 37° C., and then thetransfection mixture was aspirated and replaced with 500 μl of completemedia. Using this protocol, no significant toxicity due to transfectionwas observed.

[0099] In cells that received the PNP or control plasmids, the media waschanged two days after transfection and MeP-dR(6-methylpurine-deoxyriboside) added to the appropriate wells to a finalconcentration of 30 μg/ml. Four days later, the cells were fed by addingfresh media with MeP-dR (30 μg/ml) to the wells without removing the oldmedia. Two days later (day 6), the cells were washed once with PBS,resuspended, and counted in a 20% solution of trypan blue reagent(Trypan Blue Stain 0.4%, Gibco-BRL, Gaithersburg, Md.) using ahemacytometer.

[0100] Both T-84 colon carcinoma cells and Mel-1 melanoma cells weretransfected using DOTAP/DOPE liposomes (FIG. 3) with the SV-PNPconstruct, in which the constitutive SV40 early promoter is operablylinked to the bacterial PNP gene; or the Tyr-PNP, in which the melanomaspecific tyrosinase promoter is operably linked to the bacterial PNPgene; or the Tyr-Luc (see above); or not transfected with anyrecombinant construct (“no txf”). Only melanoma cells (Mel-1)transfected with the Tyr-PNP construct were susceptible to killing uponadministration of the prodrug MeP-dR purine analog nucleoside asdemonstrated by comparing FIG. 3A, transfected T-84 colon carcinomacells, with FIG. 3B, transfected Mel-1 melanoma cells. In contrast, whenthe constitutive SV40 early promoter was operably linked to thebacterial PNP gene (SV-PNP construct), both T-84 colon carcinoma andMel-l melanoma cells transfected with the SV-PNP construct weresusceptible to killing upon administration of the prodrug MeP-dR. Theseresults demonstrate that transcriptional targeting of the expression ofa purine analog nucleoside cleavage gene permits selective killing ofspecific tumor cells. Cell death under these conditions correlates withthe amount of MeP generated by the action of recombinant E. coli PNP onMeP-dR. The transfection of plasmid containing either a cytoplasmic or anuclear targeted β-galactosidase gene under the same conditionsindicated a low transfection efficiency (<0.1% of cells positive forLacZ).

EXAMPLE 12 Method for Identifying Candidate Prodrugs for Bacterial PNP

[0101] The following method is useful to identify substrates (prodrugs)that are cleaved more efficiently by the bacterial PNP than by mammalianPNP. Prodrugs identified by this method can then be further assessed byanimal studies for determination of toxicity, suitability foradministration with various pharmaceutical carriers, and otherpharmacological properties.

[0102] The method quantitatively measures the cleavage of substrates invitro. The purine analog nucleosides (0.1 or 1.0 mM) were incubated in500 μl of 100 mM HEPES, pH 7.4, 50 mM potassium phosphate, and with 100μg/ml E. coli PNP or 0.1 unit/ml human PNP. The reaction mixtures wereincubated at 25° C. for 1 hour, and the reactions stopped by boilingeach sample for 2 minutes. The cleavage of [¹⁴C]inosine by each enzymewas determined as a positive control. Each sample was analyzed byreverse phase HPLC to measure conversion from substrate to product. Thenucleoside and purine analogs were eluted from a Spherisorb ODSI (5 μm)column (Keystone Scientific, Inc., State College, Pa.) with a solventcontaining 50 mM ammonium dihydrogen phosphate (95%) and products weredetected by their absorbance at 254 nm, and were identified by comparingtheir retention times and absorption spectra with authentic samples.

[0103] By this analysis, MeP-dR, 2-F-dAdo, 1-deaza-2-amino-6-Cl-purine-riboside, 2-F-50-deoxyadenosine,2-Cl-20-deoxyadenosine were all shown to be good substrates forbacterial PNP and poor substrates for the mammalian PNP, and thus arepreferred candidate prodrugs which are eligible for further assessmentfor use in the methods and compositions described herein to treatmalignancies (MeP-dR is a suitable prodrug, as noted above). Substrates50-amino-50-deoxyadenosine, F-araA, and α-adenosine were moderatesubstrates for bacterial PNP and poor substrates for the mammalian PNP.Substrates xylosyl methylpurine, 2-Cl-20-F-20-deoxyadenosine,2-F-20-F-20-deoxyadenosine, and 7-ribosyl-6-mercaptopurine were poorsubstrates for both enzymes, and therefore would not be candidateprodrugs in conjunction with unmodified E. coli PNP. Similarly,substrates 7-ribosylhypoxanthine and thioguanosine were moderate to goodsubstrates for both enzymes and also would not be candidate prodrugs fortreating tumors using the compositions and methods described herein.

[0104] 2-F-dAdo and F-araA have demonstrated antitumor activity notrelated to the production of fluoroadenine. Therefore, in methodsdescribed herein, the antitumor activity of these two substrates islikely to be potentiated by metabolism by the E. coli PNP. In addition,the metabolism and toxicity of these two agents can be prevented byincubation in the presence of 2′-deoxycytidine.

[0105] Thus, by combining these substrates with 2′-deoxycytidine,antitumor activity related only to the production of fluoroadenine ispossible. TABLE II Screening of nucleotides as substrates for E. coilPNP Percent of substrate cleaved by: E. coli PNP Human PNP substrate 100μM 1 mM 100 μM 1 mM I. Nucleosides that are good substrates for E. coliPNP, but are at best poor substrates for human PNP. MeP-dR 93(87)29(24)0 0 91(86)45(21) 0(86) 0(47) FdAdo 56(69)14(18)0(70) 0(30) 60(86)38(21)0(86) 0(47) 1-deaza-2-amino- 62(87)16(23) 0(88) 0(52) 6-Cl-purine-41(86)15(21) 0(86) 0(47) riboside 2-F-5′-deoxy- 81(86)30(21) 0(88) 0(50)adenosine 65(86)44(21) 0(86) 0(47) 2-Cl-2′-deoxy- 41(86)- 0(87) —adenosine 7-ribosyl-3- 88(91*) 67(43*) 0(0*) 0(0*) deazaguanine 84(90*)83(39*) 0(95*) 0(43**) #80(85**) — 0(87**) — 7-ribosyl-6- 0 0 0 0mercaptopurine**** 0(86) 0(21) 0(86**) 0(47) 500 μM #45(65**) 35(16**)0(87**) 0.37(40**) #10(85**) 0(87**) II. Nucleosides that are moderatesubstrates for E. coli PNP, but are at best poor substrates for humanPNP. 5′-amino-5′-deoxy- 5(86) 1(19) 0(89) 0(53) adenosine 9(86) 5(21)0(86) 0(47) #29(85**) — 0(87**) — F-araA 3(86) 3(21) 0(88) 0(50) 5(86)12(21) 0(86) 0(47) α-adeno 0(86) 0(21) 0(88) 0(50) sine 3(86) 2(21)0(86) 0(47) #0(85**) — 0(87**) — III. Nucleosides that are at best poorsubstrates for both enzymes. xylosylmethyl- 0(86) 0(21) 0(88) 0(50)purine 0(86) 0(21) 0(86) 0(47) xylosyl adenine 0(78) — 0(81) — 1(56) —0(82) — 2-Cl-2′-F-2′-deoxy- 0(86) 0(21) 0(88) 0(50) adenosine 0(86)0(21) 0(86) 0(47) 2-F-2′-F-2′-deoxy- 0(86) 0(21) 0(88) 0(50) adenosine0(86) 0(21) 0(86) 0(47) 2′,3′-dideoxy 1.6 (64**) 0(15**) 1.2(85**)0.2(49**) adenosine*** #0(85**) — 0(87**) — 2′,3′-dideoxy- 2.7(64**)3(15**) 1.1(85**) 2.4(49**) inosine*** #0(85**) — 0(87**) — 3′-deoxy0(62**) 0(16**) 0(87**) 0(45**) adenosine #0(85**) — 0(87**) —5′-carboxamide #1.2(78) — 0(81) — of adenosine 0.1(56) — 0(82) —Isopropylidine #1(78) — 0(81) — of the 0(56) — 0(82) — 5′-carboxamide ofadenosine IV. Nucleosides that are substrates for both enzymes.7-ribosyl-hypo- 16(86)30(21)3(86) 5(47) xanthine 49(86)38(21)73(86)73(47) thioguanosine 49(86)38(21)73(86) 48(47)

[0106] In Table II, above, each of the numbers represent the percentconversion of the purine analog nucleoside by the phosphorylaseindicated. The numbers in parentheses are percent conversion of theinosine to hypoxanthine in the same experiment. “*” indicates thatMeP-dR was used as the control agent in place of inosine. “**” indicatesthat 6-thioguanosine was used as a positive control in place of inosine.“***” indicates questionable activity. “****” indicates that the assaywas sensitive to boiling. “#” indicates that these assays wereterminated by filtering and not by boiling.

EXAMPLE 13 In Vivo Treatment with Bacterial PNP and MeP-dR

[0107] The utility of the bacterial PNP and prodrugs such as MeP-dR toinhibit cancer growth in vivo was demonstrated in mice engrafted withtumors expressing the bacterial PNP gene. The first step required theproduction of a recombinant retrovirus containing a constitutivelyexpressed bacterial PNP gene, as described above. The bacterial PNPencoding sequence was excised from the SV/PNP plasmid and ligated bystandard techniques into the pLNSX vector. The resulting vector, pLN/PNPused the SV40 early promoter to constitutively direct the bacterialtranscription. This plasmid vector was transfected into the ψ2 packagingcell line. The supernatant collected from these cells 48 hours later wasused to infect additional ψ2 packaging cells. Twenty-four hours later,the cells were replated at a lower density (1:5- 1:10) in mediacontaining G418 in order to select for clones containing the retroviralsequences. Several clones were selected and titers of clones determinedby standard techniques. A clone with the highest titer was selected asthe source of recombinant, LN/PNP virus, and used to infect tumor cells.

[0108] The murine mammary carcinoma cell line, 16/C, was modified toconstitutively express the bacterial PNP by infection with the LN/PNPvirus. The 16/C cells were plated at a subconfluent density, and theLN/PNP virus contained within the supernatant from the ψ2-producer linewas applied in the presence of polybrene (5 μg/ml) for several hours.The media was changed to normal media for 24 hours, after which thecells were enzymatically detached and plated at a lower density in mediacontaining G418 (1 gm/L) to select infected cells. A polyclonal mixtureof G418 resistant cells, to be referred to here as “16/C-PNP cells”, wasamplified in number for engraftment into mice. Further description ofthe methods for generation of stable PNP expressing tumor cell lines isalso provided below.

[0109] Athymic (nude) mice were engrafted with the 16/C-PNP cells. Eachmouse received 2×106 cells subcutaneously (sq) in the left flank onday 1. The results are shown in FIG. 4. Control animals (n=4) weremaintained under normal nude mouse conditions that resulted inmeasurable tumors by day 13. The tumors in all of the control micecontinued to increase in size through day 29 following engraftment. Theearly treatment group (n=4) was treated by intraperitoneal (IP)injections of 6-MeP-dR at 100 mg/kg, a dose just below the maximumtolerated dose, each day for the first 4 days (days 1-4). One of thesemice was sacrificed at day 8 to study tumor histology, and two more diedat day 20, from undetermined causes, possibly due to the very highlevels of prodrug administered. Importantly, none of the mice had anydetectable tumor up to 18 days post engraftment. One mouse developed avery small tumor at day 22. The late treatment group (n=4) was treatedby intraperitoneal injections of 6-MeP-dR at 100 mg/kg each day on days13, 14, and 15 post engraftment. All of the late treatment group hadtumors of comparable size to the controls on day 13. Unlike thecontrols, the tumors in the late treatment group did not increase insize after day 15. All of these animals survived for the completeexperiment. These results clearly show that the combination of thebacterial PNP plus prodrug causes a reduction in tumor growth in vivo.

EXAMPLE 14 Generation of Stable-cell Lines Expressing E. coli PNP

[0110] High level bystander killing of cancer cells in vitro wasevaluated using stable, PNP expressing cell lines. The E. coli PNP genewas cloned into the Hind III and Stu I sites of LNSX, a retroviralvector (Miller et al., Biotechniques 7:980-990 (1989)) in which theneomycin resistance gene is LTR-driven, and the SV40 early promoterregulates E. coli PNP expression. Cloning was accomplished by excisingthe E. coli PNP gene from SV-PNP and directionally cloning the fragmentinto LNSX (Sorscher et al., Gene Ther., 1:233-238 (1994)). The constructwas then transfected using the Lipofectin reagent (Gibco BRL) into anecotropic 3T3-based packaging cell line (ψ2). In order to obtain ahigher retroviral titer, supernatants from the initial viral collectionwere used to transduce fresh ψ2 cells. Fresh medium and G418 (Gibco BRL)were added every 3 days. Producer cells capable of releasing 10⁴-10⁵infectious particles/ml growth medium were obtained, and used totransduce murine melanoma (B16), murine breast carcinoma (16/C), andhuman glioma (D54) cell lines. Three days following addition of virus,transduced cells were selected with G418 as above.

EXAMPLE 15 Cloning of the Human Tyrosinase Promoter Region andConstruction of Luciferase Reporter Vectors

[0111] Two polymerase chain reaction primers,(GATCGCTAGCGGGCTG-AAGACAATCTCTCTCTGC (SEQ ID NO. 6) andGATCGCTAGCTTCCTCTA GTCCTCACAA-GGTCT (SEQ ID NO. 7)) were used to definethe 529 base pairs (bp) of the human tyrosinase promoter immediatelyupstream of the start of translation (−451 to +78) and to incorporateNhe I sites (underlined) at both 5′ and 3′ ends of the desired product(Giebel et al., Genomics, 9:435-45 (1991); Kikuc et al., Biochem.Biophys. Acta., 1009:283-6 (1989)). Template DNA was prepared from wholehuman blood as described by Sorscher et al., Lancet, 337:1115-8 (1991).After 30 cycles of amplification, a single PCR product of the predictedsize (553 base pairs) was obtained (94° C.×1 min, denaturation, 50° C.×2minutes annealing, and 72° C.×3 minutes elongation) using I ng template,100 ng of each primer in a 100 μl reaction mixture containing 2.5 unitsTaq polymerase, 200 mM of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3),1.5 mM MgCl₂, and 0.01 % gelatin (weight/vol.). This product wasextracted with phenol/chloroform, precipitated with ethanol, digestedwith Nhe 1, and gel purified. A luciferase reporter gene vector lackingany promoter (pGL2 Basic vector, Promega) was cut with Nhe I and theabove PCR product was ligated immediately upstream of the luciferasegene. Recombinants were screened by restriction mapping and a correctlyoriented clone was identified (Tyr-Luc). A plasmid with the tyrosinasepromoter in reverse orientation (Rev-Tyr-Luc), for use as a negativecontrol, was also selected.

EXAMPLE 16 Cancer Cell Lines for Studying Gene Activation by theTyrosinase Promoter

[0112] B16 and 16/C. are of murine origin and were a gift of Dr. W.Waud, Southern Research Institute, Birmingham, Ala.; all other celllines are of human derivation. Mel-1 (melanoma) was provided by T.Carey, University of Michigan as UMCC-Mel-1. Mel-21 (melanoma) wasprovided by M. B. Khazaeli, University of Alabama, Birmingham. GP6F2(prostate) was a gift of M. Moore, Grady Memorial Hospital, Atlanta, Ga.U-373 and D54 (glioma) were provided by Yancey Gillespie, University ofAlabama, Birmingham. HeLa (cervical carcinoma), Hep G2 (hepatocellularcarcinoma), and T-84 (colon carcinoma) were obtained from the AmericanType Culture Collection. Mel-1, Mel-21, Hep G2, and HeLa cells werecultured in Earle's minimal essential medium containing Earle's salts,and 1% L-glutamine (Gibco-BRL), with 10% fetal bovine serum and 1%nonessential amino acids. T-84 and GP6F2 cells were cultured in a 1:1mixture of Dulbecco's modified Eagle's medium and nutrient mixture F-12(Ham's ) (Gibco-BRL) with 15 mM HEPES, 1% L-glutamine, and 10% fetalbovine serum. B 16, 16/C and RPMI 8226 cells were cultured in RPMImedium 1640 with 1% L-glutamine (Gibco-BRL) and 10% fetal bovine serum.All cells were cultured at 37° C. with 85% humidity and 5% CO₂.

EXAMPLE 17 Luciferase and X-gal Assays

[0113] Each plate was washed three times with PBS and 100 μl of lysisbuffer (Luciferase Assay System, Promega) was added to each well of asix-well plate. After 15 minute incubation at 37° C., the lysate andcell debris were collected. Forty μl of the lysate was added to 100 μlof luciferase assay substrate (Promega) in a clear polystyrene 12×75 mmtube, immediately placed in a luminometer (Analytical LuminescenceLaboratory model 2010) and light production measured for 15 seconds.X-gal staining for transfection efficiency using LacZ constructs was asdescribed by Sorscher el al., Gene Ther., 1:233-238 (1994).

EXAMPLE 18 Killing and Proliferation Assays

[0114] In some studies, cellular toxicity (percentage of dead cells) wasmeasured by LDH release from dying cells (Promega, CytotoxJ 96 kit). Theproliferation assay (living cell number/well) was performed using ameasurement of tetrazonium conversion to formazin during cell growth(Cell TiterJ 96 kit). Since these two assays are designed to studyapproximately 10,000 cells per condition (using 96 well trays),measurements of bystander effects below approximately 1% (100 transducedcells) were effectively limited by difficulty in accurately countingvery small numbers of transduced, viable cells.

EXAMPLE 19 Implantation of Tumor Cells Into Mice

[0115] Transduced 16/C cells were implanted in mice by subcutaneousinjection of approximately 10⁶ cells harvested from the cultures ofstably transduced 16/C cells described above. The mice were examinedvisibly for tumor growth and those with developing tumors weremaintained. To prepare mice for use in the in vivo experiments, thetumors were removed from mice with significant tumor growth and cut into30-60 mg pieces. One 30-60 mg piece of the tumor was subcutaneouslyimplanted into the subaxillary region of each female B6C3F1 mouse. Thetumors were allowed to develop and mice with tumors of 100 mm³ wereused.

[0116] For studies conducted with nu/nu mice, cells obtained from stablecultures of transduced cell lines were injected subcutaneously into theright or left flank of the mice. Mice with visible tumor growth wereused for further studies. For the administration of purine prodrug, micewere administered MeP-dR or F-araAMP by IP injection.

EXAMPLE 20 Bystander Killing by Cell Lines Expressing E. coli PNP

[0117] Transient E. coli PNP expression in a human colonic carcinomacell line is capable of mediating total cell population killing in vitroeven when only approximately 1% of cells express the E. coli PNP gene(FIG. 1). The growth characteristics of wild type and transduced B16cells, and wild type and transduced 16c cells were the same in theabsence of MeP-dR. In FIG. 5, a dose of MeP-dR (20 μg/ml) which is nottoxic to untransduced B16 melanoma or 16/C breast cancer cells was addedto mixed cultures containing an increasing population of transduced E.coli PNP expressing cells. Effects on both cell proliferation and cellsurvival were evaluated in the presence or absence of MeP-dR. In theseexperiments, concentrations of MeP-dR which had no effect onuntransduced (wild type) B16 (FIG. 5A) or 16/C. (FIG. 5B) tumor cellscompletely eliminated cell proliferation even when as few as 2% of cellsin culture expressed the E. coli PNP gene. Based upon an LDH releaseassay, total population cell killing required that 10% of B16 cells and#1% of 16/C cells expressed the PNP gene. When E. coli PNP activity inthe transduced B16 and 16/C cells was assayed by direct enzymaticmeasurement using cell free extracts, the activity measured intransduced 16/C cells was approximately 4 fold higher than in B16 cells.(16/C:10.7 nmoles MeP-dR converted/mg cell protein/hr (n=6); B16: 2.4nmoles MeP-dR converted/mg cell protein/hour (n=2); background activityin non-transduced 16/C and B 16 cells was 0 (n=4 measurements for eachcell line)).

EXAMPLE 21 Killing of Malignant Cells in Vivo: Growth of 16/C MouseBreast Carcinoma in B6C3F1 Mice

[0118] Six mice (B6C3F1) per group with established wild type 16/Ctumors were treated with an aqueous control solution, MeP-dR (100 mg/kgIP qd×3d) or 2-fluoro-arabinofuranosyladenine monophosphate (F-araAMP)(100 mg/kg IP, 5 id×3d). The wild type tumors grew rapidly in thepresence or absence of either of the prodrugs. In addition, there was nostatistically significant delay in tumor growth attributable to eitherprodrug. (Table III, Wild-type 16/C treatment). This demonstrated thatthe prodrug was not toxic to the mice at the doses given and had noeffect on non-PNP expressing tumor cells.

[0119] Six mice per group with established PNP-transduced 16/C tumorswere treated with aqueous control solution, MeP-dR or F-ara AMP, asabove. Control solution treated tumors grew rapidly, comparable to therate of growth observed with the wild type tumors. Complete tumorregression was observed in three of six in the MeP-dR treated group. Inaddition, a statistically significant delay in the time necessary forthree tumor doublings was noted for the MeP-dR treated group (p<0.01)and the F-araAMP treated group (p<0.01). (Table III, 16/C-PNPtreatment.) TABLE III Effect of MeP-dR and F-araAMP on the growth ofwild-type 16/C tumors and 16/C tumors transduced with the E. coli PNPgene (B6C3F1) Complete Days for tumor Dose/day** Regressions/Nonspecific to double 3 Day delay Treatment (mg/kg) Total Deaths/Totaltimes * mean/SD (Treated-control) Wild-type 16/C Vehicle — 0/6 — 6.2 ∀3.7   — MeP-dR 100 0/6 0/6   8.6 ∀ 0.7   2.4 F-araAMP 500 0/6 0/6   8.9∀ 2.0   2.7 16/C-PNP Vehicle — 0/6 — 8.8 ∀ 1.1   — MeP-dR 100 3/62/6**** 14.2 ∀ 3.2*** 5.4 F-araAMP 500 0/6   0/6 12.1 + 1.6*** 3.3#intervals) for three days. #significantly different from the growthrate of vehicle-treated 16/C-PNP tumors.

EXAMPLE 22 Immunological Clearance of Tumors

[0120] To demonstrate that the efficacy shown above was not due toimmune response and clearance of PNP-expressing tumors, immune deficientmice (nu/nu) were studied using a similar protocol. Four to five nude(nu/nu) mice per group were inoculated with wild type murine breastcarcinoma cells (16/C cell line), or PNP transduced 16/C cells. Micewith established tumors (approximately 100 mm³) were treated with MeP-dR(100 mg/kg/d IP×3 d) or F-araAMP (100 mg/kg/ IP 3 id×3 d).

[0121] Wild type tumors grew rapidly following either vehicle or prodrugadministration (FIG. 6). Animals with PNP-transduced tumors which weretreated with F-araAMP for three days demonstrated evidence of growthdelay for at least ten days, FIG. 7. Animals treated with MeP-dR showedsubstantial antitumor effects whether treated at a time when tumors wereestablished (days 13-15) or immediately following tumor cellsinoculations (days 1-4), FIG. 4.

EXAMPLE 23 In Vivo Activity of MeP-dR Against Human Glioma Transducedwith E. coli PNP

[0122] Female athymic nude mice (nu/nu) were implanted sc with 2×10⁷cells of either D54 parental tumor cells (D54-wt) or D54 tumor cellsthat had been transduced with the E. coli PNP (D54-PNP). After thetumors had grown to approximately 150 mg, they were treated ip witheither vehicle or 67 mg/kg of MeP-dR (IP) once a day for 3 days (days 6,7 and 8 after implantation). The tumor sizes were measured twice a weekafter treatment.

[0123] There were 6 of 6 complete regressions in mice with the D54-PNPtumors that were treated with MeP-dR (Table IV). Four of these animalshad no detectable tumors at the termination of the experiment. MeP-dRhad little effect on the D54-wt tumors. There was little or no loss ofweight in the animals that were treated with 67 mg/kg of MeP-dR,regardless of tumor implanted. Animals were followed for a total of 65days. No treated animals died in these experiments (FIGS. 8 and 9).TABLE IV Effect of MeP-dR on the growth of wild-type D54 tumors and D54tumors transduced with the E. coli PNP gene. Regressions NonspecificDoubling Days Delay Tumor-free Treatment Complete Partial Deaths/Totaltime (T-C) Survival Wild-type D54 Vehicle — — — 14 — 0/10 MeP-dR (67)0/6 0/6 1/6 21  7 0/6  D54-PNP Vehicle — — — 17 — 0/10 MeP-dR (67) 6/60/6 0/6 >56   >39   4/6 

[0124] A confirmation experiment was set up exactly as described above,except that animals were treated with two doses of MeP-dR (45 and 67mg/kg) (FIGS. 10 and 11). The results of this experiment were similar(Table V). There were 8 of 10 complete regressions in mice bearing theD54-PNP tumors that were treated with 67 mg/kg of MeP-dR. In 4 mice thetumors subsequently returned and grew. There were still 4 of 10tumor-free survivors 60 days after the treatment had stopped. Treatmentwith 45 mg/kg MeP-dR also had a marked affect on mice bearing theD54-PNP tumors. There were 2 of 10 complete regressions and 3 partialresponses. There were no tumor-free survivors in animals bearing theD54-PNP tumor that were treated with 45 mg/kg MeP-dR. Again, there wereno partial of complete remissions in animals bearing the D54 wild-typetumors treated with either 45 or 67 mg/kg of MeP-dR. The delay in thetime required to double twice due to treatment with MeP-dR was 5 to 6days in the non-transduced tumors and greater than 24 days in transducedtumors. In this experiment, the growth rate of the D54-PNP tumors wasconsiderably slower than it was in the first experiment. There was nochange in the growth rate of the D54-wt tumors. The reason for the slowgrowth rate of the D54-PNP tumors in this experiment is not known. TheFigures shown (FIGS. 8-11) only describe the growth of tumors that didnot show complete regression. (In other words, if a tumor was too smallto measure, it was not included in the average size). This means thatthe overall tumor regressions in the D54 PNP group are actually muchmore pronounced than they appear in FIGS. 8-11. TABLE V Effect of MeP-dRon the growth of wild-type D54 tumors and D54 tumors transduced with theE. coli PNP gene. Regressions Nonspecific Doubling Days Delay Tumor-freeTreatment Complete Partial Deaths/Total time (T-C) Survival Wild-typeD54 Vehicle — — — 12 — 0/10 MeP-dR (45) 0/10 0/10 0/10 17  5 0/10 MeP-dR(67) 0/10 0/10 0/10 18  6 0/10 D54-PNP Vehicle — — — 30 — 0/10 MeP-dR(45) 2/10 3/10 0/10 >54   >24   0/10 MeP-dR (67) 8/10 1/101/10 >55   >25   4/10

[0125] These results show that it is possible to cure animals thatgenerate MeP from MeP-dR at the site of the tumor without killing theanimal. This is important because MeP is a toxic agent and these resultsalleviate the concern that doses sufficient to destroy the tumor wouldrelease an amount of MeP into the body that would kill the animal.Therefore, these results indicate that the MeP released fromPNP-expressing tumors is diluted by body fluids to concentrations belowa toxic level. The gene therapy methodology of the present invention,therefore, offers a new way to generate highly toxic chemotherapeuticdrugs within a growing tumor, in such a way as to completely eliminatethe tumor without undue weight loss or other apparent toxicity. Takentogether, the present invention demonstrates the usefulness of a newclass of antitumor agents to treat of breast, melanoma, glioma, andother refractory solid tumor types in vivo.

[0126] Other additional in vivo experiments indicate that: (1) verylarge pre-existing tumors (approximately 1 gram in size) transduced withE. coli PNP show impressive regression when treated with 67 mg/kg ofMeP-dR (ip, qd×3 d); (2) F-araAMP, a clinical useful drug in human,leads to in vivo regression of PNP transduced tumors in mice; (3)2-F-2′-deoxyadenosine can be given to mice in doses similar to MeP-dRwithout toxicity, and mediates strong anti-tumor effects, equivalent toor above those seen with MeP-dR. This suggests that2-F-2′-deoxyadenosine should be a useful prodrug in vivo, since theliberated toxin, 2-F-Ade, is 10 to 100 fold more toxic than MeP.

EXAMPLE 24 Other Prodrugs

[0127] In addition to MeP-dR, two other prodrugs suitable for E. coliPNP activation in tumor cells can be applied to the methodology of thepresent invention. These prodrugs are F-araA and 2-F-2′deoxyadenosine.Both show high level killing of PNP-transduced tumor cells in vitro. Adose of 2-F-2′-deoxyadenosine (100 μM) was defined in the presence of 1mM deoxycytidine that kills cells transduced with the E. coli PNP evenwhen as few as 1% of the tumor cells express the gene. As desired, thisdose had no effect on control untransduced, tumor cells. A dose ofF-araA (500 ng/ml) also was identified that specifically killedtransduced, but not untransduced, tumor cells.

[0128] In addition, 21 purine nucleoside analogs were evaluated assubstrates for E. coli PNP by an independent protocol (Table II). Theseresults have identified 5 compounds as possible prodrugs in thisstrategy; MeP-dR, 2-F-2′-deoxyadenosine, 1deaza-2-amino-6-Cl-purine-riboside, 7-ribosyl-3-deazaguanine, and7-ribosyl-6-mercaptopurine. All of these compounds have the followingcharacteristics: the nucleoside analog is relatively nontoxic whencompared to the base of which it is composed, the nucleosides are goodsubstrates for the E. coli PNP, and they are poor substrates for thehuman PNP. Three agents that were poorly cleaved by the E. coli PNP butwere not cleaved by the human enzyme were 5′-amino-5′-deoxyadenosine,2-F-arabinofuranosyl-adenine, and α-adenosine. Compounds that were poorsubstrates for both the human and E. coli PNP were also identified.These compounds include xylosyl methylpurine, 2′,3′-dideoxyadenosine,3′-deoxyadenosine, 5′-carboxamide of adenosine, and the isopropylidineof the 5′-carboxamide of adenosine.

[0129] Kinetic constants for the cleavage of inosine, MeP-dR, F-dAdo,and F-araA by E. coli PNP were determined from enzymes isolated fromeither transduced human cells or E. coli cell pellets (Table VI). Theresults of these experiments indicated that there were little or nodifferences between the prokaryotic E. coli PNP enzyme in bacteria andafter tumor cell expression of the recombinant enzyme. In addition, itwas clear that inosine, MeP-dR, and F-dAdo were similar as substratesfor recombinant and natural E. coli PNP. F-araA was poorly cleaved by E.coli PNP with K_(m) of 543 μM and V_(max) of 1.9 mmole/mg/minute. TABLEVI Kinetic constants of MeP-dR, F-dAdo, and F-araA with E. coli PNP Sub.Source K_(m)(μM) V_(max) V_(max)/K_(m) Inosine Bacteria 46 132 2.9 D54cells — — — MeP-dR Bacteria 68 251 3.7 D54 cells 107 5.4 0.050 F-dAdoBacteria 44 190 4.3 D54 cells 38 2.1 0.056 F-araA Bacteria 543 1.90.0034 D54 cells 510 0.023 0.000043

EXAMPLE 25 Recombinant E. coli that Express 100-Fold More PNP ActivityThan Wild-Type E. coli and Use of this Bacterium to Deliver E. coli PNPto Tumor Cells

[0130] Previous studies showed that MeP-dR, F-araAMP, and F-dAdo havegood activity against D54 glioma tumors expressing the E. coli PNP gene.MeP-dR, F-araAMP, and F-dAdo were less active against tumors composed ofmixtures of wild-type tumor cells and transduced tumor cells at a ratioof 80 to 20, respectively. This result indicated that increasedexpression of E. coli PNP in the tumor cell may be necessary todemonstrate in vivo bystander activity with these three compounds. Theamount of expression of E. coli PNP activity in the transduced D54tumors cells was between 200 to 300 nmoles of MeP-dR cleaved per mgprotein per hour.

[0131] In an effort to increase the amount of E. coli PNP expressed intumor cells and to develop a vector to realistically deliver E. coli PNPto tumors in animals, E. coli was transformed with the E. coli PNP gene(SEQ ID No: 5) and created a recombinant E. coli that expressed veryhigh levels of E. coli PNP, approximately 1,000,000 nmoles of MeP-dRcleaved per mg protein per hour. In order to accomplish this, a plasmidcapable of mediating high level expression of E. coli PNP wasconstructed by excising the E. coli PNP (SEQ ID No: 5) from a transfervector (pTM-1 PNP) by a double restriction enzyme digestion with Nco Iand Xho I. (see FIG. 12). pTRC His B (Invitrogen, Carlsbad, Calif.) wasdigested with Nco I and Xho I, and the E. coli PNP fragments describedabove were directionally ligated into pTRC so as to initiate E. coli PNPtranslation from the first methionine. The ligation reaction was used totransform competent E. coli by standard techniques. In this case, theDH5% strain was used, but other strains of E. coli or other bacteriacould be used for the same purpose. Recombinants were selected onampicillin, and correct orientation and integration of the insert wasverified by restriction mapping. The plasmid is designed to allowfurther induction of E. coli PNP activity after treatment withisopropyl-%-D-thio-galactopyranoside (IPTG), and in some experimentsthis induction was verified as additional evidence of the predictedbehavior of the recombinant plasmid. FIG. 13 shows a protein band thatis present in the recombinant E. coli (Lanes 10, 11, 15) that isinducible with IPTG (Lanes 15′, 11′, and 10′). No protein was detectedin the wild-type strains at this position, or in strains expressing aninducible control protein (WT NBD2 and WT NBD2′). The amount of E. coliPNP in wild-type bacteria was approximately 10,000 nmoles of MeP-dRcleaved per mg protein per hour. Therefore, this recombinant E. coli had100-fold more E. coli PNP activity than wild-type cells.

[0132] Tumors in the flanks of mice were injected with this recombinantE. coli, and the activity of E. coli PNP in the tumors was determinedthirty minutes and forty-eight hours after injection of bacteria. Afterthirty minutes the amount of E. Coli PNP activity in the tumors wasapproximately 100,000 nmoles of MeP-dR cleaved per mg protein per hour,whereas at forty-eight hours the activity in the tumors had increasedmodestly to approximately 200,000 nmoles of MeP-dR cleaved per mgprotein per hour (see Table VII). These results indicated thatapproximately 1000 times more E. coli PNP could be delivered to tumorcells than was present in the D54-PNP tumors. This result verifies thathuman patients could be treated by inoculating their tumors with thisrecombinant bacteria. TABLE VII E. coli PNP activity in tumors injectedwith recombinant E. coli Sample 30 minutes 40 hours 1-1 0.1 ml/tumor100,000 >223,000 -2 >178,000 >133,000 -3 78,000 >212,000 2-1 0.2ml/tumor >223,000 >227,000 -2 >184,000 >222,000 -3 >179,000 >292,000

[0133] >indicates that there was considerable cleavage of MeP-dR (>40%)at the earliest time measured (fifteen minutes). There was little or nocleavage at zero (0) time. The specific activity was determined fromthis number which was not in the linear portion of the activity curve.Therefore, these numbers are a slight underestimate of the trueactivity.

EXAMPLE 26 Treatment of Lewis Lung Tumors with E. coli and MeP-dR

[0134] Subcutaneous Lewis Lung tumors on the flanks of mice(approximately 300 mg) were injected with E. coli bacteria transfectedwith E. coli purine nucleoside phosphorylase gene (plasmid pTRCPNP)containing SEQ ID No: 5. Mice were treated with 0, 16.8, 33.5, or 67mg/kg of MeP-dR once a day for three days, and the tumor size wasmonitored over the following 18 days. The control consisted ofsaline/Tween 80. This initial experiment was designed for two purposes.First, to determine whether E. coli over-expressing the PNP gene couldbe given in combination with MeP-dR and without undue toxicity. Second,to evaluate anti-tumor effects in this particular animal model andstrain of mouse. E. coli PNP activity was measured thirty minutes andforty-eight hours after injection of bacteria in representative LewisLung tumors injected with bacteria but not treated with MeP-dR. The PNPactivity was 16,000 and 28,000 nmoles of MeP-dR cleaved per mg proteinper hour at 0.5 and forty-eight hours, respectively (each number is theaverage of two determinations). The anti-tumor results (shown in FIG.14) indicated that treatment with E. coli bacteria that express E. coliPNP activity plus MeP-dR slowed the growth of these fast growing tumors.In this experiment, treatment with 33.5 mg/kg of MeP-dR delayed tumorgrowth by approximately 42% without host toxicity. Treatment with ahigher dose of MeP-dR (67 mg/kg, IP Qd×3d) was toxic, with severaldeaths due to the combined therapy. Nevertheless, this treatment arm wasinformative, since anti-tumor effects were again observed. These resultsindicated that E. coli bacteria could deliver significant amounts of E.coli PNP to tumor cells in an animal and that this enzyme could activateMeP-dR resulting in an antitumor response.

EXAMPLE 27 Injection of Methyl Purine (MeP) into Established HumanPancreatic Tumors

[0135] CF PAC (human pancreatic adenocarcinoma) cells were grown in T75flasks, trypsinized, washed with PBS and then inoculated subcutaneouslyinto female SCID mice at approximately 2×10⁷ cells per animal. Thetumors were allowed to grow for several days until they reached the sizeof approximately 80-100 millimeters. At this point, groups of fiveanimals were treated either with vehicle (PBS), PBS containing methylpurine (1.67 mg/kg) or PBS with a higher dose of methyl purine (5.0mg/kg). Drug or vehicle was injected intratumorally with 3-4 needlepasses. Animals were treated with either vehicle or drug one time eachday for three days. The growth of the tumors was followed. Measurementsin two dimension were used to approximate the overall tumor size. Tumorstreated at the higher dose of MeP were completely arrested in terms oftheir growth, while the lower dose of MeP led to an intermediate effectupon tumor growth. The results (see FIGS. 15 and 16) indicated that MePhas a very potent anti-tumor effect when injected directly into tumorsand further support the notion that methyl purine and derivativesthereof is an effective anti-tumor agent, either after direct tumorinoculation or when generated within a tumor by virtue of expression ofthe E. coli PNP gene. The results also suggest that direct inter-turmoalinjection of cytotoxic purine analogs or other cytotoxic drugs canelicit profound anti-tumor effects in vivo. For example, purine analogswhich are known to have strong cytotoxic activity such as2-fluoroadenine, 2-fluoroadenosine, 9- [ribosyl]-6-methylpurine,2-amino-6-chloro-1-deazapurine, 3 -deazaguanine, 6-thioguanine, and6-mercaptopurine can be directly administered to a tumor to causeregression and/or inhibit tumor growth.

Summary

[0136] The following data summarizes in vitro and in vivo experiments inwhich the efficacy of the claimed delivery vehicles and methods werefurther demonstrated. Experiments to show the killing of cancer cells invitro used mixed populations of PNP expressing and nonexpressing cells.The results demonstrated that a small population of PNP expressing cellscan facilitate the death of large numbers of surrounding, non-PNPexpressing cells. In vivo efficacy was demonstrated by implanting intomice transduced and nontransduced tumor cells. Tumor size decreased inmice implanted with PNP-transduced tumor cells upon the administrationof a prodrug purine analog. The results indicate that the claimedmethods are applicable to the treatment of mammalian malignantdisorders.

[0137] The results provided were generated using art recognized in vivoand in vitro models of mammalian malignancy. These results demonstratethat: (1) a small number of PNP expressing tumor cells can facilitate inthe killing of surrounding, non-PNP expressing cells, (2) PNP expressioncan be controlled in a tissue specific fashion, (3) the claimedtherapeutic method works with a variety of tumor types in art recognizedmodels of mammalian malignancy, and (4) that purine analogs such asmethyl purine can be used to inhibit tumor growth.

[0138] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0139] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentmethods, procedures, treatments, molecules, and specific compoundsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

1 7 1 37 DNA Escherichia coli 1 gatcgcggcc gcatggctac cccacacatt aatgcag37 2 45 DNA Escherichia coli 2 gtacgcggcc gcttactctt tatcgcccagcagaacggat tccag 45 3 36 DNA Escherichia coli 3 gatcgctagc gggctctgaagacaatctct ctctgc 36 4 35 DNA Escherichia coli 4 gatcgctagc tcttcctctagtcctcacaa ggtct 35 5 5013 DNA Escherichia coli 5 gtttgacagc ttatcatcgactgcacggtg caccaatgct tctggcgtca ggcagccatc 60 ggaagctgtg gtatggctgtgcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120 gcactcccgt tctggataatgttttttgcg ccgacatcat aacggttctg gcaaatattc 180 tgaaatgagc tgttgacaattaatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240 taacaatttc acacaggaaacagcgccgct gagaaaaagc gaagcggcac tgctctttaa 300 caatttatca gacaatctgtgtgggcactc gaccggaatt atcgattaac tttattatta 360 aaaattaaag aggtatatattaatgtatcg attaaataag gaggaataaa ccatggctac 420 cccacacatt aatgcagaaatgggcgattt cgctgacgta gttttgatgc caggcgaccc 480 gctgcgtgcg aagtatattgctgaaacttt ccttgaagat gcccgtgaag tgaacaacgt 540 tcgcggtatg ctgggcttcaccggtactta caaaggccgc aaaatttccg taatgggtca 600 cggtatgggt atcccgtcctgctccatcta caccaaagaa ctgatcaccg atttcggcgt 660 gaagaaaatt atccgcgtgggttcctgtgg cgcagttctg ccgcacgtaa aactgcgcga 720 cgtcgttatc ggtatgggtgcctgcaccga ttccaaagtt aaccgcatcc gttttaaaga 780 ccatgacttt gccgctatcgctgacttcga catggtgcgt aacgcagtag atgcagctaa 840 agcactgggt attgatgctcgcgtgggtaa cctgttctcc gctgacctgt tctactctcc 900 ggacggcgaa atgttcgacgtgatggaaaa atacggcatt ctcggcgtgg aaatggaagc 960 ggctggtatc tacggcgtcgctgcagaatt tggcgcgaaa gccctgacca tctgcaccgt 1020 atctgaccac atccgcactcacgagcagac cactgccgct gagcgtcaga ctaccttcaa 1080 cgacatgatc aaaatcgcactggaatccgt tctgctgggc gataaagagt aaagagtaaa 1140 tcgatggcct gaattcgaagcttggctgtt ttggcggatg agagaagatt ttcagcctga 1200 tacagattaa atcagaacgcagaagcggtc tgataaaaca gaatttgcct ggcggcagta 1260 gcgcggtggt cccacctgaccccatgccga actcagaagt gaaacgccgt agcgccgatg 1320 gtagtgtggg gtctccccatgcgagagtag ggaactgcca ggcatcaaat aaaacgaaag 1380 gctcagtcga aagactgggcctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg 1440 agtaggacaa atccgccgggagcggatttg aacgttgcga agcaacggcc cggagggtgg 1500 cgggcaggac gcccgccataaactgccagg catcaaatta agcagaaggc catcctgacg 1560 gatggccttt ttgcgtttctacaaactctt tttgtttatt tttctaaata cattcaaata 1620 tgtatccgct catgagacaataaccctgat aaatgcttca ataatattga aaaaggaaga 1680 gtatgagtat tcaacatttccgtgtcgccc ttattccctt ttttgcggca ttttgccttc 1740 ctgtttttgc tcacccagaaacgctggtga aagtaaaaga tgctgaagat cagttgggtg 1800 cacgagtggg ttacatcgaactggatctca acagcggtaa gatccttgag agttttcgcc 1860 ccgaagaacg ttttccaatgatgagcactt ttaaagttct gctatgtggc gcggtattat 1920 cccgtgttga cgccgggcaagagcaactcg gtcgccgcat acactattct cagaatgact 1980 tggttgagta ctcaccagtcacagaaaagc atcttacgga tggcatgaca gtaagagaat 2040 tatgcagtgc tgccataaccatgagtgata acactgcggc caacttactt ctgacaacga 2100 tcggaggacc gaaggagctaaccgcttttt tgcacaacat gggggatcat gtaactcgcc 2160 ttgatcgttg ggaaccggagctgaatgaag ccataccaaa cgacgagcgt gacaccacga 2220 tgcctgtagc aatggcaacaacgttgcgca aactattaac tggcgaacta cttactctag 2280 cttcccggca acaattaatagactggatgg aggcggataa agttgcagga ccacttctgc 2340 gctcggccct tccggctggctggtttattg ctgataaatc tggagccggt gagcgtgggt 2400 ctcgcggtat cattgcagcactggggccag atggtaagcc ctcccgtatc gtagttatct 2460 acacgacggg gagtcaggcaactatggatg aacgaaatag acagatcgct gagataggtg 2520 cctcactgat taagcattggtaactgtcag accaagttta ctcatatata ctttagattg 2580 atttaaaact tcatttttaatttaaaagga tctaggtgaa gatccttttt gataatctca 2640 tgaccaaaat cccttaacgtgagttttcgt tccactgagc gtcagacccc gtagaaaaga 2700 tcaaaggatc ttcttgagatcctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 2760 aaccaccgct accagcggtggtttgtttgc cggatcaaga gctaccaact ctttttccga 2820 aggtaactgg cttcagcagagcgcagatac caaatactgt ccttctagtg tagccgtagt 2880 taggccacca cttcaagaactctgtagcac cgcctacata cctcgctctg ctaatcctgt 2940 taccagtggc tgctgccagtggcgataagt cgtgtcttac cgggttggac tcaagacgat 3000 agttaccgga taaggcgcagcggtcgggct gaacgggggg ttcgtgcaca cagcccagct 3060 tggagcgaac gacctacaccgaactgagat acctacagcg tgagctatga gaaagcgcca 3120 cgcttcccga agggagaaaggcggacaggt atccggtaag cggcagggtc ggaacaggag 3180 agcgcacgag ggagcttccagggggaaacg cctggtatct ttatagtcct gtcgggtttc 3240 gccacctctg acttgagcgtcgatttttgt gatgctcgtc aggggggcgg agcctatgga 3300 aaaacgccag caacgcggcctttttacggt tcctggcctt ttgctggcct tttgctcaca 3360 tgttctttcc tgcgttatcccctgattctg tggataaccg tattaccgcc tttgagtgag 3420 ctgataccgc tcgccgcagccgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 3480 aagagcgcct gatgcggtattttctcctta cgcatctgtg cggtatttca caccgcatat 3540 ggtgcactct cagtacaatctgctctgatg ccgcatagtt aagccagtat acactccgct 3600 atcgctacgt gactgggtcatggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc 3660 ctgacgggct tgtctgctcccggcatccgc ttacagacaa gctgtgaccg tctccgggag 3720 ctgcatgtgt cagaggttttcaccgtcatc accgaaacgc gcgaggcagc agatcaattc 3780 gcgcgcgaag gcgaagcggcatgcatttac gttgacacca tcgaatggtg caaaaccttt 3840 cgcggtatgg catgatagcgcccggaagag agtcaattca gggtggtgaa tgtgaaacca 3900 gtaacgttat acgatgtcgcagagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg 3960 gtgaaccagg ccagccacgtttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg 4020 gagctgaatt acattcccaaccgcgtggca caacaactgg cgggcaaaca gtcgttgctg 4080 attggcgttg ccacctccagtctggccctg cacgcgccgt cgcaaattgt cgcggcgatt 4140 aaatctcgcg ccgatcaactgggtgccagc gtggtggtgt cgatggtaga acgaagcggc 4200 gtcgaagcct gtaaagcggcggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc 4260 attaactatc cgctggatgaccaggatgcc attgctgtgg aagctgcctg cactaatgtt 4320 ccggcgttat ttcttgatgtctctgaccag acacccatca acagtattat tttctcccat 4380 gaagacggta cgcgactgggcgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg 4440 ctgttagcgg gcccattaagttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa 4500 tatctcactc gcaatcaaattcagccgata gcggaacggg aaggcgactg gagtgccatg 4560 tccggttttc aacaaaccatgcaaatgctg aatgagggca tcgttcccac tgcgatgctg 4620 gttgccaacg atcagatggcgctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc 4680 gttggtgcgg atatctcggtagtgggatac gacgataccg aagacagctc atgttatatc 4740 ccgccgttaa ccaccatcaaacaggatttt cgcctgctgg ggcaaaccag cgtggaccgc 4800 ttgctgcaac tctctcagggccaggcggtg aagggcaatc agctgttgcc cgtctcactg 4860 gtgaaaagaa aaaccaccctggcgcccaat acgcaaaccg cctctccccg cgcgttggcc 4920 gattcattaa tgcagctggcacgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 4980 cgcaattaat gtgagttagcgcgaattgat ctg 5013 6 34 DNA Artificial Sequence Synthetic polymerasechain reaction primer 6 gatcgctagc gggctgaaga caatctctct ctgc 34 7 33DNA Artificial Sequence Synthetic polymerase chain reaction primer 7gatcgctagc ttcctctagt cctcacaagg tct 33

1. A method of killing replicating or non-replicating, targetedmammalian cells and bystander cells, comprising the steps of: (a)delivering a purine cleavage enzyme to the targeted mammalian cells; and(b) contacting the targeted cells with an effective amount of asubstrate for the purine cleavage enzyme, wherein the substrate isnon-toxic to mammalian cells and is cleaved by the cleavage enzyme toyield a purine base which is toxic to the targeted mammalian cells andbystander cells, to kill the mammalian cells contacted with the cleavageenzyme and the bystander cells.
 2. The method of claim 1, wherein thesubstrate is selected from the group consisting of9-(β-D-2-deoxyerythropentofuranosyl)-6-methylpurine,2-amino-6-chloro-1-deazapurine riboside, 7-ribosyl-3-deazaguanine,arabinofuranosyl-2-fluoroadenine, 2-fluoro-2N-deoxyadenosine,2-fluoro-5N-deoxyadenosine, 2-chloro-2N-deoxy-adenosine,5N-amino-5N-deoxy-adenosine, α-adenosine, MeP-2N,3N-dideoxyriboside,2-F-2N,3N-dideoxyadenosine, MeP-3N-deoxyriboside, 2-F-3N-deoxyadenosine,2-F-adenine-6N-deoxy-β-D-allofuranoside, 2-F-adenine-α-L-lyxofuranoside,MeP-6N-deoxy-β-D-allofuranoside, MeP-α-L-lyxofuranoside,2-F-adenine-6N-deoxy-α-L-talofuranoside, MeP-6N-deoxy-α-L-talofuranosideand 7-ribosyl-thioguanine.
 3. The method according to claim 1, whereinthe enzyme is targeted to the cells by conjugating the enzyme to anantibody.
 4. The method according to claim 1, wherein the enzyme isselected from the group consisting of a natural non-human PNP, amodified non-human PNP, a natural non-human hydrolase, a modifiednon-human hydrolase, a natural human PNP, a modified human PNP, anatural human hydrolase, a modified human hydrolase.
 5. The methodaccording to claim 1, wherein the targeted cells are selected from thegroup consisting of: tumor cells and virally infected cells.
 6. Acomposition for killing targeted mammalian cells, comprising: (a) anenzyme that cleaves a purine substrate; and (b) an effective amount ofthe purine substrate to kill the targeted cells when cleaved by theenzyme.
 7. The composition of claim 6, wherein the substrate is selectedfrom the group consisting of9-(β-D-2-deoxyerythropentofuranosyl)-6-methylpurine,2-amino-6-chloro-1-deazapurine riboside, 7-ribosyl-3-deazaguanine,arabinofuranosyl-2-fluoroadenine, 2-fluoro-2N-deoxyadenosine,2-fluoro-5N-deoxyadenosine, 2-chloro-2N-deoxy-adenosine,5N-amino-5N-deoxy-adenosine, α-adenosine, MeP-2N,3N-dideoxyriboside,2-F-2N,3N-dideoxyadenosine, MeP-3N-deoxyriboside, 2-F-3N-deoxyadenosine,2-F-adenine-6N-deoxy-β-D-allofuranoside, 2-F-adenine-α-L-lyxofuranoside,MeP-6N-deoxy-β-D-alofuranoside, MeP-α-L-lyxofuranoside,2-F-adenine-6N-deoxy-α-L-talofuranoside, MeP-6N-deoxy-α-L-talofuranosideand 7-ribosyl-thioguanine.
 8. The composition of claim 6, wherein theenzyme is selected from the group consisting of purine nucleosidephosphorylase or hydrolase.
 5. The composition according to claim 6,wherein the targeted cells are selected from the group consisting of:tumor cells and virally infected cells.